Sustained suppression of SHIV89.6P replication in macaques by vaccine-induced CD8+ memory T cells
Yin, Jiangmeia; Dai, Anlana; Kutzler, Michele Ab; Shen, Andingc; LeCureux, Jonathanc; Lewis, Mark Gd; Waldmann, Thomase; Weiner, David Ba; Boyer, Jean Da
aDepartment of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, USA
bCollege of Medicine, Drexel University, Philadelphia, Pennsylvania, USA
cDepartment of Biology, Calvin College, Grand Rapids, Michigan, USA
dResearch Section, Bioqual, Rockville, USA
eMetabolism Branch, National Cancer Institute, Frederick, Maryland, USA.
Received 9 May, 2008
Revised 24 June, 2008
Accepted 27 June, 2008
Correspondence to Jean D. Boyer, Department of Pathology and Laboratory Medicine, 505 SCL, 422 Curie Boulevard, Philadelphia, PA 19104, USA. Tel: +1 215 662 2382; fax: +1 215 573 9436; e-mail: firstname.lastname@example.org
Objective: We previously demonstrated that a strategy of co-immunizing cynomologous macaques with a simian/human immunodeficiency virus DNA-based vaccine and a plasmid encoding macaque interleukin (IL)-15 induces a strong CD8+ and CD4+ effector T-cell response that, upon subsequent challenge with SHIV89.6P, controls viral replication and protects immunized animals against ongoing infection. In this follow-up study, we measured viral replication 2 years after vaccination challenge and determined the mechanism by which antigen-specific CD8+ T cells suppress viral replication.
Method: From the original group of 18, we assessed the immune response in the 13 surviving animals. In addition, using cM-T807, we depleted CD8 lymphocytes to assess the role CD8 cells play in suppression of viral replication.
Result: We found that peripheral blood mononuclear cells from vaccinated animals had a robust simian immunodeficiency virus Gag-specific IFN-γ response. In addition, in the DNA and IL-15 group, we observed higher levels of simian immunodeficiency virus Gag-specific, proliferating CD8+ T cells. The profile of these cells revealed more central memory than effector cells. When we transiently depleted animals of CD8+ T cells, plasma viral load increased, and peak viral load was lower in the DNA and IL-15 group compared with the DNA alone and control groups. As CD8+ T cells recovered, viral replication was controlled and we observed an increase in the number of antigen-specific effector CD8+ T cells.
Conclusion: We conclude that co-immunization with a simian/human immunodeficiency virus DNA-based vaccine and IL-15 achieves sustained viral suppression and that vaccine-induced CD8+ memory T cells, which differentiate into effector cells, are central to that suppression.
Tremendous effort has been devoted to the development of an HIV vaccine that elicits a strong cellular immune response. Cellular immune responses, specifically CD8+ T-cell responses, play a major role in controlling HIV and simian immunodeficiency virus infection (SIV) [1–4]. In acute HIV and SIV infection, the emergence of an HIV-specific CD8+ T-cell response correlates well with decline in viremia [1,5–7]. In chronic HIV infection, high levels of HIV-specific polyfunctional CD8+ T cells, which are present in nonprogressors, are a positive prognostic sign [8,9]. The strongest evidence that CD8+ T cells control HIV replication comes from a macaque model. SIV-infected macaques depleted of CD8+ T cells are unable to control viral replication and exhibit dramatic increases in plasma viral load [10–12]. In aggregate, these observations suggest that induction of CD8+ T-cell responses will be a critical component of an effective HIV vaccine.
Memory T cells have more recently gained a great deal of attention with regard to protective immunity against HIV and SIV infection. Compared with memory CD4+ T cells, memory CD8+ T cells appear to play a dominant role in the antiviral response. Therefore, induction of functional memory T cells should also be an important goal of HIV vaccine development. Memory T cells can be divided into two subsets based on surface marker expression and function: central memory T (TCM) cells and effector memory T (TEM) cells. TCMs readily differentiate into effector cells and are home to T-cell areas of secondary lymphoid organs, but have little or no effector function. In contrast, TEMs are characterized by rapid effector function, producing high levels of IFN-γ and perforin within hours of antigenic stimulation [13–16].
In a previous study, we showed that co-immunization of cynomologous macaques with an SIV gag DNA-based vaccine and a plasmid encoding macaque IL-15 induces vaccine-specific, IFN-γ-producing, CD8+ and CD4+ effector T cells that suppress viral replication . However, the duration of viral suppression after vaccination-challenge and the mechanism by which antigen-specific CD8+ T cells control viral replication are not clear.
In this 2-year follow-up study, we demonstrate that co-immunization with an SIV gag DNA vaccine and IL-15 achieves sustained viral suppression, elicits robust CD8+ and CD4+ T-cell responses, and induces central memory T cells. We further demonstrate that, in response to viral rebound caused by transient CD8+ T-cell depletion, CD8+ memory T cells differentiate into effector cells and control viral replication.
Materials and methods
Macaques were housed at the Bioqual in Rockville, Maryland, USA. These facilities are accredited by the American Association for the Accreditation of Laboratory Animal Care International and meet National Institutes of Health standards as set forth in the Guidelines for Care and Use of Laboratory Animals. The University of Pennsylvania Institutional Animal Care and Use Committee (IACUC) reviewed and approved all procedures carried out by Bioqual.
Immunization and challenge
Macaques were immunized and challenged as previously described . In brief, macaques were immunized three times intramuscularly with buffer, 2 mg of pSIVgag DNA, or 2 mg of pSIVgag DNA coinjected with 2 mg of pmacIL-15. After 84 weeks, the second series of immunizations was given, which included an increase in dose to 3 mg of pSIVgag and pmacIL-15 and incorporated 3 mg of pSIVpol and pHIVenv. Macaques were challenged with 300 monkey infectious doses (MIDs) by the intravenous route with SHIV89.6P (kindly provided by Norman Letvin, Harvard University, Cambridge, Massachusetts, USA) 11 weeks after the final boost.
Peptides used in this study were obtained from the National Institutes of Health AIDS Reagent and Reference Reagent Program (NIH Repository, Bethesda, Maryland, USA). The peptides that were used corresponded to the complete sequences of SIVmac239 Gag (n = 125), SIVmac239 Pol (n = 263), or HIV-1 Env (n = 211). Peptides were 15 amino acids in length with 11 amino acid overlap. Peptides were resuspended in dimethyl sulfoxide (DMSO) at a final concentration of approximately 100 mg/ml and divided into pools. Gag was divided into three pools (peptides 1–41 for pool 1, 42–83 for pool 2, and 84–125 for pool 3), Env was divided into four pools (peptides 1–53 for pool 1, 54–106 for pool 2, 107–159 for pool 3, and 160–211 for pool 4), and Pol was divided into five pools (peptides 1–53 for pool 1, 54–106 for pool 2, 107–158 for pool 3, 159–210 for pool 4, and 211–263 for pool 5), which were labeled utilizing consecutive numbers designated by the NIH Repository. Gag mix, Pol mix, and Env mix comprised all of the peptides corresponding to the complete sequence of SIVmac239 Gag, Pol, and HIV-1 Env, respectively. Peptides were stored at −20°C until use.
Viral load in plasma and CD4+ T cells
Plasma viral load was quantitated using a procedure described by Matano et al. . The assay has a threshold sensitivity of 40 RNA copies/ml of plasma. The latent reservoir in CD4+ T cells was determined by p27 enzyme-linked immunosorbent assay (ELISA) as previously described . In brief, CD4+CD25− T lymphocytes were isolated from fresh peripheral blood mononuclear cells (PBMCs) using Miltenyi beads and LS columns. The isolated lymphocytes were incubated with CEM-174 cells in limiting dilutions to activate and amplify latent virus. The supernatants were sampled after 14 days and tested for p27 by ELISA (ZeptoMetrix). In addition, DNA was isolated from the CD4+CD25− T cells and viral DNA was detected by nested PCR with the outer primers (5′-GA TTT GGA TTA GCA GAA AGC C-3′ and 5′-CT GCA TGT AGT TCT TTA GCA GAT CC-3′) and inner primers (5′-CCA TTA GTG CCA ACA GGC TCA G-3′ and 5′-CA TGG GGA AAT TGC GGG GCT TC-3′).
ELISpot assay for IFN-γ and perforin
ELISpot assays using IFN-γ and perforin reagents (MabTech, Sweden) and nitrocellulose plates (Millipore, Billerica, Massachusetts, USA) were performed according to the manufacturer's instructions. A positive response was defined as greater than 50 spot-forming cells (SFCs) per million PBMCs and two times above background. Each sample was performed in triplicate with peptides.
T-cell proliferation and memory T-cell subset assay
PBMCs were incubated with carboxyfluorescein succinimidyl ester (CFSE) (5 μmol/l) for 8 min at 37°C. Cells were washed and incubated with antigens (SIVp27/gag peptide mix) at a concentration of 5 μg/ml for 5 days at 37°C in 96-well plates. Cultures without Gag peptide were used to determine the background proliferative responses. PBMCs were immunostained with the following mAbs: anti-CD3 APC-Cy7 (BD-Pharmingen, San Diego, California, USA), anti-CD4 PerCP-Cy5.5 (BD-Pharmingen), anti-CD8 APC (BD-Pharmingen), anti-CD28 ECD (Beckman Coulter, Fullerton, California, USA), and anti-CD95 PE-Cy5 (BD-Pharmingen). Central memory and effector memory T cells were defined as CD28+CD95+ and CD28−CD95+, respectively . Stained cells were washed in phosphate-buffered saline (PBS) and fixed (Cell-Fix). Stained and fixed cells were acquired on an LSRI flow cytometer (BD Biosciences, Franklin Lakes, New Jersey, USA) instrument using CellQuest software (BD Biosciences) and analyzed with FlowJo software (Tree Star, Ashland, Oregon, USA).
CD8+ T-cell depletion
To deplete cynomologous macaques of CD8+ cells, animals were injected with cM-T807 (provided by the NIH Nonhuman Primate Reagent Resource Center). Specifically, each macaque was weighed and the antibody was delivered by injection according to the following schedule: 10 mg/kg subcutaneously on day 0 and 5 mg/kg intravenously on days 3, 7, and 10. CD8+ lymphocyte depletion was confirmed by demonstrating an absence of CD3+CD8+ and CD3−CD8+ lymphocytes by flow cytometry. The antibodies used for the flow cytometry were anti-CD4 FITC (clone L200; BD-Pharmingen), anti-CD8 PE (clone DK25; Dako, Glostrup, Denmark), and anti-CD3 PE-Cy5 (clone SP34; BD-Pharmingen).
Sustained suppression of viral replication in macaques co-immunized with simian immunodeficiency virus gag DNA and IL-15
Using the simian/human immunodeficiency virus (SHIV) and cynomologous macaque model, we previously examined the efficacy of and the cellular immune profile induced by a vaccine strategy that immunized animals with an SIV gag DNA-based vaccine alone or in combination with a plasmid encoding IL-15 . In that study, macaques were immunized three times intramuscularly with buffer (control group), 2 mg of pSIVgag DNA (DNA group), or 2 mg of pSIVgag DNA coinjected with 2 mg of pmacIL-15 (DNA and IL-15 group). After 84 weeks, a second series of immunizations was performed, which included both an increase in dose of pSIVgag and pmacIL-15 to 3 mg as well as the addition of 3 mg of pSIVpol and pHIVenv. Eleven weeks after the final injection, all animals were challenged intravenously with 300 MIDs of SHIV89.6p. The results of that study demonstrated that co-immunization with SIV gag DNA and IL-15 induced a cellular immune profile that suppressed SHIV89.6 replication .
One objective of this study, which followed the three groups of vaccinated-challenged animals for 2 years, was to determine whether suppression of viral replication was sustained. During the 2-year period of observation, four of the six macaques in the control group developed AIDS-like syndrome and died and one of the six macaques in the DNA group failed to control viral replication (data not shown). In contrast, the remaining 13 macaques controlled viral replication and plasma viral load remained below detectable limits by viral RNA quantitation (Table 1, left column). In addition to measuring plasma viral load 2 years after vaccination challenge, we assayed for replication competent, latent virus in CD4+CD25− T cells by p27 ELISA. We detected virus in CD4+CD25− T cells in three of the 13 monkeys, including two in the control group and one in the DNA group (Table 1, middle column). When animals were assessed for the presence of viral DNA in CD4+ T cells, all macaques were found to be positive (Table 1, right column). Taken together, these results show that, in spite of the fact that virus is not completely cleared from CD4+ T cells, immunization with SIV gag DNA and IL-15 induces sustained suppression of viral replication.
Robust IFN-γ response to simian immunodeficiency virus gag in macaques co-immunized with DNA and IL-15
To explore the mechanism of sustained viral suppression in macaques in the DNA and IL-15 group, we assessed the IFN-γ response to SIV Gag. PBMCs were obtained from macaques that controlled viral replication, the cells were stimulated for 24 h with pools of SIV Gag, SIV Pol, and HIV Env peptides, and the IFN-γ response was measured by an ELISA-linked immunospot (ELISpot) assay. In the control group, the average number of IFN-γ-producing cells specific to SIV Gag was 659 SFCs per million PBMCs (Fig. 1a). By comparison, in the vaccine groups, an enhanced SIV Gag-specific IFN-γ response was observed. The average number of IFN-γ-producing cells per million PBMCs was 1412 SFCs in the DNA group and 1441 SFCs in the DNA and IL-15 group (Fig. 1a). When we assayed the IFN-γ response to stimulation with HIV Env (Fig. 1b) or SIV Pol (Fig. 1c) peptides, no difference was detected between the three groups. Although having only two animals in the control group prevented formal statistical analysis, a summary of the IFN-γ response data (Fig. 1d) showed a trend toward a greater number of SIV Gag-specific lymphocytes in the vaccine groups compared with the control group.
Higher proliferative capacity in T cells from co-immunized animals
We next investigated the ability of SIV Gag-specific CD8+ and CD4+ effector cells to proliferate after antigen stimulation. PBMCs isolated from macaques 2 years after vaccination challenge were incubated with CFSE, washed, and stimulated for 5 days with growth media, SIV Gag peptides, or concanavalin A (ConA). Following stimulation, T-cell proliferation was measured by flow cytometry. We found that the proliferative responses of CD4+ cells (Fig. 2a) and CD8+ cells (Fig. 2b) were higher in the DNA and IL-15 group than in the control or DNA groups. Furthermore, the average proliferative responses were higher in CD8+ T cells (7.34%) (Fig. 2d) than in CD4+ T cells (2.11%) (Fig. 2c). These results demonstrate that the proliferative capacity of CD8+ T cells is significantly enhanced in macaques co-immunized with the DNA vaccine and IL-15.
Higher frequency of central memory T cells in proliferating T cells
We then compared the frequency of proliferating memory T-cell subsets among the three groups of macaques 2 years after vaccination challenge. Proliferating memory T cells were divided into two subsets by surface marker expression: central memory T cells (TCM) were defined as CD28+CD95+ and effector memory T cells (TEM) were defined as CD28−CD95+  (Fig. 3a). When we evaluated the frequency of TCMs and TEMs in proliferating CD4+ cells (Fig. 3b, upper panels), we detected a significantly higher percentage of TCMs in the DNA and IL-15 group (average 30.6%) compared with the DNA alone (average 5%) and control (average 2.2%) groups (Fig. 3b, upper left panel). Among the three groups, there was no difference in the percentages of TEMs (Fig. 3b, upper right panel).
We also evaluated the frequency of TCM and TEM in proliferating CD8+ T cells. The percentages of both TCM and TEM were higher in the DNA and IL-15 group than in the DNA and control groups (Fig. 3b, lower panels), although only the TCM difference reached statistical significance. An average of 10.8% TCM and 2.1% TEM were detected in proliferating CD8+ T cells isolated from animals in the DNA and IL-15 group (Fig. 3b, lower panels). Taken together, the data show that co-immunization with SIV gag DNA and IL-15 results in more TCM in both proliferating CD4+ and CD8+ T cells as well as higher proliferating TEM in CD8+ T cells.
Co-immunization protects against depletion of total CD4+ and CCR5+CD4+ T cells from mucosal sites
In unvaccinated, uninfected macaques, more than 50% of the CD4+ T cells in mucosal sites are CCR5+ [21–23]. Depletion of CD4+ T cells from mucosal sites is a hallmark of HIV and SIV infection. Furthermore, studies have demonstrated almost complete depletion of the CCR5+ subset of CD4+ T cells from mucosal sites in HIV-infected humans and SIV-infected rhesus macaques [21–24].
We were interested in determining whether or not immunization provides protection against depletion of total CD4+ T cells or the CCR5+ subset or both. To address these questions, we performed ileal biopsies of macaques in the control, DNA alone, and DNA and IL-15 groups at 2 years after vaccination challenge, isolated lymphocytes, and determined the percentage of CD4+ T cells and CCR5+CD4+ T cells in biopsied tissue by flow cytometry. We found the highest percentage of CD4+ T cells in macaques that had been co-immunized with DNA and IL-15 and the lowest percentage of CD4+ T cells in the control group (data not shown). We further found that, although all animals displayed some degree of depletion of CCR5+CD4+ T cells at 2 years after vaccination challenge, only 0.7% of CD3+ T cells were CD4+CCR5+ in the control group, 1.0% of CD3+ T cells were CD4+CCR5+ in the DNA alone group, and 2.7% of CD3+ T cells were CD4+CCR5+ in the DNA and IL-15 group (data not shown). Taken together, these results suggest that co-immunization provides some protection against the depletion of both total CD4+ T cells and the CCR5+ subset of CD4+ T cells.
CD8+ T cells play a major role in sustained suppression of viral replication
To evaluate the role of CD8+ T cells in sustained suppression of viral replication, we depleted CD8+ T cells in animals that had successfully controlled SHIV89.6 replication and then assessed plasma viral load. To deplete CD8+ T cells, animals were injected intravenously with the mouse–human chimeric anti-CD8 monoclonal antibody, cM-T807 . At various time points after antibody treatment, both the number of CD8+ T cells in peripheral blood and plasma viral load were determined. After 3 days of antibody treatment, the number of CD8+ T cells in peripheral blood, on an average, reduced by 95% and rebounded to pretreatment levels by day 28 (Fig. 4a). Concomitant with the depletion of CD8+ T cells in peripheral blood, plasma viral load increased in most animals (Fig. 4b). Notably, the peak viral load in animals co-immunized with DNA and IL-15 was 0.8 log lower and occurred up to 7 days later than the peak viral load in animals vaccinated with DNA alone (Fig. 4b). These findings support the hypothesis that CD8+ T cells play a major role in controlling viral replication. In addition, these findings suggest that co-immunization with SIV gag DNA and IL-15 results in stronger viral suppression than immunization with DNA alone.
Enhanced CD8+ T-cell effector function following viral rebound
Next, we examined the mechanism by which CD8+ T cells from vaccinated animals controlled viral replication following a transient increase in plasma viral load. To do this, we assessed both the IFN-γ response and perforin secretion in animals at day 28 after CD8+ T-cell depletion. At this time point, CD8+ T cells had recovered and were at or near baseline levels prior to depletion (Fig. 4a) and plasma viral load had decreased from the levels during rebound (Fig. 4b). When we measured SIV Gag-specific secretion of IFN-γ and perforin by ELISpot assay on day 28 after depletion, we found that the vaccine groups had higher numbers of SIV Gag-specific, IFN-γ-producing cells than the control group (Fig. 5a). The average number of effector cells per million PBMCs was 2607 SFCs in the DNA and IL-15 group, 3144 SFCs in the DNA alone group, and 1188 SFCs in the control group (Fig. 5b). When we compared the IFN-γ response to SIV Gag before and after CD8+ T-cell depletion, we found that in each group the response was greater after CD8+ T-cell depletion (Fig. 5b). There was no significant difference between the three groups in the IFN-γ responses to SIV Pol and HIV Env (data not shown).
When we assayed perforin secretion, we found that animals in the DNA and IL-15 group secreted more perforin in an SIV Gag-specific than the control and DNA alone groups (Fig. 5c). The average number of SFCs per million PBMCs was 438 in the DNA and IL-15 group, 233 in the DNA alone group, and 144 in the control group (Fig. 5d). When we compared SIV Gag-specific secretion of perforin before and after CD8+ T-cell depletion, we found that the perforin response, like the IFN-γ response, was greater after CD8+ T-cell depletion (Fig. 5d). Together, these results suggest that animals in the DNA and IL-15 group have enhanced CD8+ T-cell effector function following viral rebound.
In this 2-year follow-up study, we demonstrated that co-immunization of cynomologous macaques with an SIV Gag DNA-based vaccine and a plasmid encoding macaque IL-15 achieves sustained suppression of SHIV89.6p replication. We further demonstrated that the vaccine-induced, CD8+ effector T-cell immune response is central to that suppression. Effective cellular immune responses protected co-immunized animals from SHIV89.6p, as indicated by low plasma viral loads (Table 1) and high levels of CD4+ and CD4+CCR5+ T cells in mucosal tissue (data not shown). Sustained viral suppression correlated with the ability of effector T cells to proliferate secrete IFN-γ upon stimulation with SIV Gag (Fig. 1). Using IL-15 as an adjuvant to the DNA-based vaccine resulted in improved proliferative capacity of CD8+ T cells (Fig. 2) and induction of greater numbers of TCMs (Fig. 3). After transient CD8+ T-cell depletion, plasma viral levels increased in most animals, demonstrating the central role of CD8+ T cells in viral suppression (Fig. 4). Following this rebound in plasma viral load, CD8+ memory T cells differentiated into effector cells and again controlled viral replication (Fig. 5). In animals co-immunized with SIV Gag DNA and IL-15, these effector functions after CD8+ T-cell depletion and recovery were even stronger than before depletion (Fig. 5).
IL-15, a cytokine that is important for the maintenance of long-lasting, high-avidity T-cell responses to invading pathogens, achieves this by supporting the proliferation and survival of CD8+ memory T cells [25–34]. Because of these properties, IL-15 is often used in vaccine and therapeutic platforms. IL-15 as an adjuvant enhances the function and longevity of CD8+ T cells in mice . Treating PBMCs obtained from HIV-infected patients with IL-15 enhances anti-HIV immune function . IL-15 enhances survival and function of HIV-specific CD8+ T cells in vitro . Recently, IL-15 treatment during acute SIV infection was shown to elicit strong SIV-specific CD8+ T-cell responses in vivo .
In our study, consistent with our previous work , animals in the DNA and IL-15 group had lower viral loads in plasma and in CD4+CD25− T cells than animals in the DNA alone and control groups (Table 1). By comparison, some studies indicate that IL-15 increases the viral set point after SIV or HIV infection [19,38]. The studies report that IL-15 abrogates vaccine-induced decreases in plasma viral load in SIVmac251-infected macaques. These functional differences have considerable implications for the incorporation of cytokines into molecular vaccines against HIV [32–34].
In the field of HIV vaccine development, immunological protection mechanisms are being investigated both to guide vaccine design and define clinical trial endpoints. Some investigators propose that protection correlates with the persistence of CD4+ central memory T cells [15,39]. Others suggest that protection correlates with CD8+ T-cell responses, specifically Gag-specific CD8+ central memory T-cell responses . Nevertheless, it is agreed that an effective vaccine against HIV must induce protective central memory T cells . Our findings demonstrate that co-immunization of cynomologous macaques with a SHIV DNA-based vaccine and IL-15 achieves sustained viral suppression. Moreover, our study begins to define the mechanism by which antigen-specific CD8+ T cells suppress viral replication.
This research was supported in part by National Institutes of Health (NIH) Grants N01-AI-50010, P01-A1-071739, R01-A1-071186, and the National Institutes of Health Intramural Research Program. J.M., A.D., A.S., J.L., and M.G.L. all performed research and generated the data presented. M.A.K., T.W., and D.B.W. provided reagents and guidance. J.D.B. designed experiments and oversaw the project and writing of the paper.
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