In vivo replication kinetics of a nef-deleted strain of HIV-1
Crowe, Suzanne Ma,b; Ho, David Dc; Marriott, Debbied; Brew, Bruced; Gorry, Paul Ra,b; Sullivan, John Se,f; Learmont, Jennye; Mills, Johna,b
aMacfarlane Burnet Institute for Medical Research and Public Health, Melbourne, Australia
bDepartment of Medicine, Monash University, Melbourne, Australia
cAaron Diamond AIDS Research Centre, the Rockefeller University, New York, USA
dSt Vincents Hospital, Darlinghurst, NSW, Australia
eAustralian Red Cross Blood Service, Sydney, NSW, Australia
fFaculty of Medicine, The University of Sydney, Sydney, NSW, Australia.
Received 8 July, 2004
Revised 4 January, 2005
Accepted 28 January, 2005
The subject of this study (donor D36) is a member of the Sydney Blood Bank Cohort (SBBC) and is infected with an attenuated strain of HIV-1 [1,2]. He was infected in early 1981, and when studied had been infected for 18 years. Regular monitoring showed stable CD4 lymphocyte counts within the normal range (median over 13.8 years 504 cells/μl) and low viral loads (median 2850 HIV-1-RNA copies/ml) without antiretroviral therapy [1,2], until approximately one year before the study when his CD4 cell count began to decline and his viral load rose to between 10 000 and 20 000 HIV-1-RNA copies/ml . He subsequently became ill with HIV encephalopathy [cerebrospinal fluid (CSF) viral load > 750 000 copies/ml] requiring hospitalization, despite continuing infection only with nef-deleted HIV-1 . In January 1999 treatment commenced with zidovudine, abacavir and nevirapine.
Blood was taken for baseline viral load and CD4 lymphocyte counts on two occasions one week apart before antiretroviral therapy, on initiation of therapy, then at 6, 12, 18, 21, 24 h, then daily for the first 6 days, then every 3–4 days to 33 days, then intermittently thereafter. Viral load was performed using the Quantiplex branched DNA 3.0 assay (Bayer Diagnostics, Emeryville, CA, USA); CD4 lymphocytes were enumerated by dual-platform flow cytometry.
The kinetics of viral load decline were performed according to previously published methods [4,5].
Approximately 24 h after the initiation of antiretroviral therapy the viral load decreased with an initial slope (first phase; delta) of 0.18 per day (T1/2 of 3.7 days; line A, Fig. 1). The second phase decay had a slope (mu) of 0.053 per day (T1/2 of 13 days; line B, Fig. 1). Overall, the viral load fell from 4000 copies/ml (log10 3.6) at the commencement of therapy to 50 copies/ml (log10 1.7) by day 29 (Fig. 1).
The CD4 lymphocyte count rose from 210 (10%) to 304 cells/μl (16%) by 29 days after commencing therapy (Fig. 1). Three months after the initiation of therapy plasma and CSF viral loads were both 50 copies/ml or less and his CD4 cell count had risen to 384 cells/μl.
This is the first study on the in-vivo replication kinetics of an attenuated strain of HIV-1, in this instance caused by deletions in the nef gene and deletions and rearrangements in the long-terminal repeat . The first-phase slope of decline of HIV RNA in this SBBC member was 0.18/day (T1/2 of 3.7 days), slower than that seen in all previously studied individuals infected with wild-type HIV-1 after the commencement of antiretroviral therapy [4–7].
The apparently prolonged in-vivo half-life of nef-deleted virions could be explained by a longer intrinsic in-vivo half-life of lymphocytes productively infected with nef-deleted virus compared with wild-type HIV-1, consistent with some in-vitro data  but not all. Individual variation is an unlikely explanation as the slope is outside the range reported for 48 individuals with wild-type HIV . First-phase decay rates are reportedly influenced by the initial viral load and CD4 cell count , but D36 had a low baseline viral load, which should have accelerated the first-phase decay rate . It is also unlikely that the prolonged decay phase was caused by the suboptimal penetration of these antiretroviral drugs into central nervous system tissue and CSF, or to the suboptimal potency of the antiretroviral regimen.
The 24 h delay before the viral load began to decrease was surprising because nevirapine should inhibit HIV replication more rapidly in vivo than nucleoside reverse transcriptase inhibitors (which require intracellular phosphorylation) or protease inhibitors (which act late in the virus life cycle). However, a similar delay in viral load decline was also seen in all individuals infected with wild-type HIV and treated with a protease inhibitor .
The second phase of plasma HIV-1-RNA decay had a slope (mu) of 0.053/day, a T1/2 of approximately 13 days, typical for a long-lived, productively infected population of cells such as macrophages, and similar to that previously reported for wild-type HIV-1 (mean of 14.1 days) . The HIV-1 strain infecting D36 was dual-tropic and infected macrophages .
These data suggest that inhibiting the in-vivo replication of this nef-deleted strain of HIV-1 with antiretroviral therapy increases CD4 lymphocyte numbers, unequivocally documenting the pathogenicity of these strains. This conclusion is entirely consistent with long-term in-vivo studies of the SBBC  and of macaques infected with nef-deleted strains of SIV . However, our study was on a single member of the SBBC and confirmation of these data is required. If HIV-1 strains are to be attenuated sufficiently for use as vaccines, they must contain further mutations in addition to the nef gene deletion.
This study was approved by the Human Ethics Committee of the State of Victoria, and written informed consent was obtained from D36, who is thanked for his willingness to participate. A. Dunne is thanked for her assistance in performing the viral load assays.
Sponsorship: S.M. Crowe and J. Mills were supported by the National Centre in HIV Virology Research (NCHVR) through a grant from the Commonwealth Department of Health and Aged Care.
1. Deacon NJ, Tsykin A, Solomon A, Smith K, Ludford-Menting M, Hooker DJ, et al
. Genomic structure of an attenuated quasi species of HIV-1 from a blood transfusion donor and recipients. Science 1995; 270:988–991.
2. Learmont JC, Geczy AF, Mills J, Ashton LJ, Raynes-Greenow CH, Garsia RJ, et al
. Immunologic and virologic status after 14 to 18 years of infection with an attenuated strain of HIV-1. A report from the Sydney Blood Bank Cohort. N Engl J Med 1999; 340:1715–1722.
3. Churchill M, Sterjovski J, Gray L, Cowley D, Chatfield C, Learmont J, et al
. Longitudinal analysis of nef/LTR-deleted HIV-1 in blood and CSF of a long-term survivor who developed HIV-associated dementia. J Infect Dis 2004; 190:2181–2186.
4. Perelson AS, Neumann AU, Markowitz M, Leonard JM, Ho DD. HIV-1 dynamics in vivo
: virion clearance rate, infected cell life-span, and viral generation time. Science 1996; 271:1582–1586.
5. Perelson AS, Essunger P, Cao Y, Vesanun M, Hurley A, Saksela K, et al
. Decay characteristics of HIV-1-infected compartments during combination therapy. Nature 1997; 387:188–191.
6. Wei X, Ghosh SK, Taylor ME, Johnson VA, Emini EA, Deutsch P, et al
. Viral dynamics in human immunodeficiency virus type 1 infection. Nature 1995; 373:117–122.
7. Wu H, Kuritzkes DR, McClernon DR, Kessler H, Connick E, Landay A, et al
. Characterization of viral dynamics in human immunodeficiency virus type 1-infected patients treated with combination antiretroviral therapy: relationships to host factors, cellular restoration, and virologic end points. J Infect Dis 1999; 179:799–807.
8. Jekle A, Schramm B, Jayakumar P, Trautner V, Schols D, De Clercq E, et al
. Coreceptor phenotype of natural human immunodeficiency virus with nef deleted evolves in vivo
, leading to increased virulence. J Virol 2002; 76:6966–6973.
9. Hofmann-Lehmann R, Vlasak J, Williams AL, Chenine A-L, McClure HM, Anderson DC, et al
. Live attenuated, nef-deleted SIV is pathogenic in most adult macaques after prolonged observation. AIDS 2003; 17:157–166.
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