Post-exposure prophylaxis for SIV revisited: animal model for HIV prevention

Abstract

Background: A 4-week, uninterrupted treatment with 9-(2-phosphonyl-methoxypropyly)adenine (PMPA, commonly called tenofovir) completely prevents simian immunodeficiency virus (SIVmne) infection in cynomolgus macaques if treatment begins within 24 hours after SIVmne inoculation, but is less effective if treatment is delayed or duration of treatment is shortened. Critical factors for efficacy include timing and duration of treatment, potency of antiretroviral drug and a contribution from antiviral immune responses. Therefore, we evaluated the impact of one or more treatment interruptions plus SIVmne re-exposures on efficacy of PMPA treatment to prevent SIVmne infection in cynomolgus macaques. We also evaluated whether macaques with pre-existing SIV immune responses show increased efficacy of treatment. Eight PMPA-treated, virus-negative and seronegative macaques, and five PMPA-treated, virus-negative but weakly or strongly seropositive macaques were re-inoculated with SIVmne and treated with PMPA starting 24 hr post inoculation. Thereafter, they received either a 5-week treatment involving one interruption plus one SIVmne challenge or a 10-week treatment involving six interruptions plus six SIVmne challenges early during treatment. Parameters measured were plasma SIV RNA, SIV-antibody response, CD4+ T lymphocyte subsets and in vivo CD8+ cell-suppression of virus infection.

Results: All seronegative macaques developed persistent antibody response beginning 4 to 8 weeks after stopping PMPA-treatment in absence of viremia in a majority of macaques and coinciding with onset of intermittent viremia in other macaques. In contrast, all weakly or strongly seropositive macaques showed immediate increase in titers (> 1600) of SIV antibodies, even before the end of PMPA-treatment, and in absence of detectable viremia. However, in vivo CD8+-cell depletion revealed CD8 cell-suppression of viremia and persistence of virus in the macaques as long as 2 years after PMPA-treatment, even in aviremic macaques. Unlike untreated macaques, a treated macaque controlled viral replication and blocked CD4+ T cell depletion when challenged with a heterologous chimeric SIV/HIV-1 virus called SHIV89.6P.

Conclusion: A single interruption plus one SIVmne challenge was as sufficient as six interruptions plus six SIVmne challenges in reducing efficacy of PMPA, but results in long-term persistence of virus infection suppressed by CD8+ cells. Efficacy of PMPA treatment was highest in macaques with pre-existing SIV immune responses.

Figure 1

A. A schematic drawing SIV_mne inoculations and treatment regimens of different groups. Arrows show schedule of SIV_mne inoculation. The solid horizontal bar indicate schedule of daily PMPA treatment (30 mg/kg, subcutaneous). AID₅₀ is 50% animal infectious dose of the SIV_mne used in the studies. One AID₅₀ of the SIVmne stock was approximately10 50% tissue culture infectious dose (TCID₅₀) by intravenous inoculation, and 100 TCID₅₀ by intrarectal inoculation. Macaques in groups B (plus macaque 93043 in group D) received a 10-week PMPA treatment beginning 24 hours after virus inoculation, including treatment interruption plus SIV_mne re-inoculation with 10 AID₅₀ SIV_mne at weekly intervals during the first six weeks of treatment. Macaques in groups C and D received a 5-week PMPA treatment starting 24 hours after virus inoculation, including one treatment interruption plus SIV_mne re-inoculation with 10 AID₅₀ SIV_mne at week 1 of treatment. Macaque 95020 (group E) was untreated. B. The exact timing of events during the 72 hour interruption, including timing of SIV_mne re-inoculation during the treatment interruption. Treatment was initiated 24 hours after SIVmne inoculation, then continued for 5 days. Thereafter, treatment was withheld for 72 hours, during which macaques were re-inoculated with SIV_mne at 48 h and re-started on treatment at 72 h. After the last inoculation, treatment was continued uninterrupted for 28 days. Note that the during the treatment interruption, the 48-hour interval between the end of treatment and SIV_mne re-inoculation was approximately one half-life of PMPA active metabolites in resting lymphocytes or three times the half-life in activated lymphocytes [26].

Figure 2

Plasma viral load levels in untreated and PMPA-treated macaques after intravenous inoculation with uncloned SIV_mne. Group A were naïve untreated macaques. Macaques in group B (plus macaque 93043 in group D) received a 10-week PMPA treatment beginning 24 hours after virus inoculation, including treatment interruption plus SIV_mne re-inoculation at weekly intervals during the first six weeks of treatment. Macaques in groups C and D received a 5-week PMPA treatment starting 24 hours after virus inoculation, including one treatment interruption plus SIVmne re-inoculation at week 1 of treatment. SIV RNA in plasma measurements were performed at Bayer Diagnostics (Berkeley, CA) using a branched DNA (bDNA) signal amplification assay for SIV. This bDNA assay has a lower-limit of detection of125 RNA copies/ml.

Figure 3

Anti-SIV IgG antibody response in untreated and PMPA-treated macaques after intravenous inoculation with uncloned SIV_mne. Group A were naïve untreated macaques. Macaques in group B (plus macaque 93043 in group D) received a 10-week PMPA treatment beginning 24 hours after virus inoculation, including treatment interruption plus SIV_mne re-inoculation at weekly intervals during the first six weeks of treatment. Macaques in groups C and D received a 5-week PMPA treatment starting 24 hours after virus inoculation, including one treatment interruption plus SIVmne re-inoculation at week 1 of treatment. Titers are expressed as the reciprocal of the highest dilution that was positive by HIV-2 EIA (Sanofi-Pasteur, Redmond, WA). The lowest plasma dilution used was 1:40.

Figure 4

Relationship between levels of CD8+ cells in peripheral blood and plasma viral load in macaques during in vivo depletion of CD8+ lymphocytes. Depletion of CD8+ lymphocytes was achieved by using the monoclonal anti-CD8 antibody (cMT807) as described in the text. Depletion studies were performed 2.3 years after the end of PMPA-treatment. Levels of CD8+ lymphocytes were measured by flow cytometry and plasma viral load was quantitated by a branched DNA (bDNA) signal amplification assay for SIV as described in the text. Macaque 93217 and 93194 were from group C. Macaque 93040 and 93043 were from group D.

Figure 5

Plasma viral load (A) and SIV antibody response (B) in naïve and PEP-macaques inoculated intravenously with 10 AID₅₀ chimeric SIV/HIV-1 called SHIV_89.6P. PEP, post exposure prophylaxis. Macaques 95020 and M94312 were persistently virus-negative and weakly SIV-antibody positive (V^-Ab^±) after the first SIV_mne infection/PMPA PEP regimen (1^st PEP) [15]. Four years later, macaque M94312 received a second PEP regimen (2^nd PEP) involving one treatment interruption plus SIV_mne challenge at week 1 of a 5-week PMPA treatment. Thereafter, this macaque became persistently virus-negative and strongly-SIV antibody positive (V^-Ab⁺). Macaque 95020 was not given a second PEP and remained persistently virus-negative and weakly-antibody positive (V^-Ab^±). Both macaques were then challenged with SHIV_89.6P at the same time at 2.5 years after the 2^nd PEP (i.e. 6.5 years after the 1^st PEP). Two naïve macaques (99111 and 99107) served as infection controls. Plasma viremia was quantified by a branched DNA (bDNA) signal amplification assay for SIV by Bayer Diagnostics (Berkeley, CA). The target probes for the assay are designed to hybridize with the pol region of SIVmac strains of virus. This assay has a lower limit of detection of125 RNA copies/mL. Titers of SIV antibodies are expressed as the reciprocal of the highest dilution of plasma that was positive by HIV-2 EIA (Sanofi-Pasteur, Redmond, WA). The lowest plasma dilution used was 1:20.

Figure 6

Lymphocyte subsets in naïve and PEP-macaques inoculated intravenously with 10 AID₅₀ chimeric SIV/HIV-1 called SHIV_89.6P. Figure 1 shows absolute numbers of CD4 T cells. Figure 1B shows CD4:CD8 ratio. PEP, post exposure prophylaxis. Macaques 95020 and M94312 were persistently virus-negative and weakly SIV-antibody positive (V^-Ab^±) after the first SIV_mne infection/PMPA PEP regimen (1^st PEP) [15]. Four years later, macaque M94312 received a second PEP regimen (2^nd PEP) involving one treatment interruption plus SIV_mne challenge at week 1 of a 5-week PMPA treatment. Thereafter, this macaque became persistently virus-negative and strongly-SIV antibody positive (V^-Ab⁺). Macaque 95020 was not given a second PEP and remained persistently virus-negative and weakly-antibody positive (V^-Ab^±). Both macaques were then challenged with SHIV_89.6P at the same time at 2.5 years after the 2^nd PEP (i.e. 6.5 years after the 1^st PEP). Two naïve macaques (99111 and 99107) served as infection controls. Lymphocyte subsets are analyzed in peripheral blood by FACScan for absolute number of CD4+CD3+ T lymphocytes and CD8+CD3+ T lymphocytes and used to calculate the ratio of CD4:CD8 T lymphocytes in peripheral blood.