IL-15 increases the frequency of effector memory CD8+ T cells in rhesus monkeys immunized with HIV vaccine.

Abstract

Several studies have suggested that interleukin (IL)-15 is a promising adjuvant that promotes cellular immunity when administered with human immunodeficiency virus (HIV) vaccine. Here we evaluated the effect of IL-15 plasmid on HIV-specific immune responses, especially cellular immunity, in eight rhesus monkeys. These monkeys were immunized three times with HIV DNA vaccine with or without IL-15 plasmid and boosted with recombinant Tiantan strain vaccinia virus-based HIV vaccine (rTV) 22 weeks after the first immunization. Although we did not detect any significant differences in the HIV-specific CD8(+) T-cell response between monkeys with IL-15 coimmunization and monkeys with HIV vaccine alone, our results showed that the frequency of effector CD8(+) memory T cells in the peripheral blood was significantly higher in monkeys with IL-15 coimmunization than those with HIV vaccine alone at almost all of the time points examined. Furthermore, the titers of anti-HIV antibodies were higher in Group T than those in Group C after rTV boosting. These findings in rhesus monkeys suggest that IL-15 may be useful as a cytokine adjuvant for HIV vaccine.

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IL-15 increases the percentage of effector memory CD8+ T cells
in rhesus monkeys immunized with HIV vaccine
Shirui Li,
Department of Immunology, Institute of Basic Medical Sciences, Chinese Academy of Medical
Sciences and School of Basic Medicine, Peking Union Medical College, Beijing 100005, China
Xiangrong Qi,
National Institute for Viral Disease Control and Prevention, China CDC, Beijing 100052, China
Yingying Gao,
National Institute for Viral Disease Control and Prevention, China CDC, Beijing 100052, China
Yanling Hao,
Division of Virology and Immunology, National Center for AIDS/STD Control and prevention,
Chinese Center for Disease Control and Prevention, Beijing 100050, China
Lianxian Cui,
Department of Immunology, Institute of Basic Medical Sciences, Chinese Academy of Medical
Sciences and School of Basic Medicine, Peking Union Medical College, National Key Laboratory
of Medical Molecular Biology, Beijing 100005, China
Li Ruan, and
National Institute for Viral Disease Control and Prevention, China CDC, Beijing 100052, China
Wei He
Department of Immunology, Institute of Basic Medical Sciences, Chinese Academy of Medical
Sciences and School of Basic Medicine, Peking Union Medical College, National Key Laboratory
of Medical Molecular Biology, Beijing 100005, China
Abstract
Several studies suggested that interleukin (IL)-15 is a promising adjuvant of human
immunodeficiency virus (HIV) vaccine via promoting cellular immunity. Here we evaluated the
effect of IL-15 plasmid on HIV specific immune response, especially cellular immunity, in 8
rhesus monkeys. These monkeys were immunized three times with HIV DNA vaccine with or
without IL-15 plasmid, and boosted with recombinant Tiantan strain vaccinia virus-based HIV
vaccine (rTV) 22 weeks after the first immunization. As the results, the percentages of effector
CD8+ memory T cells in the peripheral blood were significantly higher in the group with IL-15
co-immunization (Group T) than those in the group with HIV vaccine alone (Group C) at almost
all the time points, while no significant difference in HIV specific CD8+ T cells response was
found between Group T and Group C throughout the experiment. The titers of anti-HIV antibodies
were higher in Group T than those in Group C after rTV boosting as well. These findings in rhesus
monkeys suggest that IL-15 may be used as a cytokine adjuvant for HIV vaccine.
Corresponding author: Wei He, Department of Immunology, Institute of Basic Medical Sciences, Chinese Academy of Medical
Sciences and School of Basic Medicine, Peking Union Medical College, National Key Laboratory of Medical Molecular Biology, 5
Dong Dan San Tiao, Beijing 100005, China, Tel: 86-10-65136981; Fax: 86-10-65105909; heweiimu@public.bta.net.cn.
NIH Public Access
Author Manuscript
Cell Mol Immunol. Author manuscript; available in PMC 2012 June 18.
Published in final edited form as:
Cell Mol Immunol. 2010 November ; 7(6): 491–494. doi:10.1038/cmi.2010.44.
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Keywords
adjuvant; effector memory CD8+ T cell; HIV vaccine; IL-15
Introduction
Since human immunodeficiency virus (HIV) was identified as the pathogen of acquired
immune deficiency syndrome (AIDS) in the 1980’s, extensive efforts have been made to
study this virus. Because cellular immune response is indispensable in antiviral immunity
(1-3), current HIV vaccines are aimed to promote cellular immunity to eradicate HIV virus.
Among these vaccines, cytokine adjuvants like interleukin-2 (IL-2), IL-12, IL-15, and IL-21
have been used to enhance the anti-HIV cellular immunity (4-7). IL-15 belongs to the 4 α-
helix bundle cytokine family, secreted by antigen presenting cells, such as dendritic cells
and monocytes, during the early stage of immune response. Its receptor shares the common
γ chain with those of interleukin (IL)-2, 3, 7, 9, and 21. Therefore, IL-15 exhibits similar
biological effects to IL-2, promoting T cell and natural killer cell proliferation (8,9).
A recent study showed that the function and survival of CD8+ T cells can be promoted in
vivo by co-immunization of optimized IL-15 plasmid and HIV vaccine after CD4+ T cells
were depleted (10). Also, HIV specific cellular and humoral immunity in mice, and the
percentage of central CD8+ memory T cells in the spleen, can be enhanced with IL-15
plasmid co-immunization (11). Here we carried out study in rhesus monkeys to observe the
effects of IL-15 plasmid on CD8+ T cells.
Materials and Methods
Vaccines
HIV vaccine is constructed by National Center for AIDS/STD Control and Prevention,
China CDC, which is a multiple epitope vaccine in the form of DNA plasmid containing the
gene of gp140, gag, pol, tat, nef, and rev, or recombinant Tiantan strain vaccinia virus (rTV)
expressing env, gag, pol of HIV strain B/C. IL-15 cDNA plasmid was constructed in our
laboratory as previously described (11).
Animal immunization
Eight rhesus monkeys, 4 male and 4 female, 3-4 years old, 4-5 kg in body weight, bred and
fed at Institute of Medical Biological Science, CAMS. The rhesus monkey immunization
and assay schedule was shown in Figure 1. Briefly, animals were immunized
intramuscularly using 4mg HIV DNA plasmid (Group C) or 4mg HIV DNA plasmid with
1mg IL-15 cDNA plasmid (Group T) on week 0, 4, and 8, respectively, and boosted with
1×107 pfu rTV on week 22. On week 6, 10, 18, 23, 26 and 30, blood from each animal was
sampled and examined. All animal studies were carried out in accordance with the standards
set forth in the Guide for the Care and Use of Laboratory Animals (published by the
National Academy of Science, National Academy Press, Washington, D.C.).
Enzyme-linked immunospot (ELISPOT) assay
Fifty microliter (μl) of properly diluted anti-interferon(IFN)-γ coating antibodies were
added into each well and incubated overnight at 4°C. 200 μl Blocking buffer B (1×) is added
and incubated for another 1 hour at 37°C. Rhesus monkey PBMCs were plated at 2×105
cells per well and stimulated for 24 h at 37°C in 5% CO2 with 5μg/ml different HIV peptide
pools (grant from NIH) or PMA (positive control). After 1 hour incubation at 37°C with 100
μl of properly diluted biotinylated IFN-γ detection antibodies, 50 μl of properly diluted
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GABA solution was added into each well and incubated for another 1 hour at 37°C. Then 30
μl of freshly prepared Activator I/II solution was added to each well and incubated at room
temperature in the dark. When clear spots developed, stop the reaction by rinsing the wells
with deionized water. The spots were counted by an immunospot image analyzer.
Antibody detection
Plates were coated with 0.2μg/well of HIV-1 env antigen for 2 hours at 37°C. After
blocking with PBS containing 10% bovine serum albumin (BSA) for 1 hour at 37°C, 100μl
of sera prepared by doubling dilution was added to each well and incubated for 1 hour at
37°C. The plates were then incubated at 37°C for 1 hour with goat anti-mouse IgG
conjugated to horseradish peroxidase at a 1:5000 dilution. The substrate solution was added
100μl/well. 10-15 minutes later, the reaction was stopped by 2M H2SO4 and reported an
optical density at 450 nm.
Flow cytometry
PBMCs were incubated for 30 minutes at 4°C with anti-human CD8-PE (RPA-T8),
CD45RA-Pecy5 (5H9) or purified anti-human CCR7 (2H4) antibody (BD Pharmigen). After
washes anti-mouse IgM was added and incubate at 4°C for 30 minutes. After washes,
samples were re-suspended with PBS and immediately analyzed on a FACSort flow
cytometer (Becton Dickinson).
Statistical analysis
Statistical analyses were performed with SPSS 11.5. Comparisons of immune responses
between 2 groups of rhesus monkeys were performed by two-tailed t tests. In all cases, P
values of <0.05 were considered significant.
Results
IL-15 increased the percentage of effector memory CD8+ T cells in peripheral blood in
rhesus monkeys
Although IFN-γ production determined by ELISPOT was not significantly increased in
rhesus monkey PBMCs from Group T compared with Group C (data not shown), changes in
portion of CD8+ T cells were noticed in the group received IL-15 plasmid co-immunization.
As shown in Figure 2, the percentages of effector CD8+ memory T cells (TEM) in the
peripheral blood in rhesus monkeys were higher in Group T than those in Group C at most
time points. In addition, the difference was statistically significant 8 weeks after rTV
boosting (P <0.05). The percentage of central CD8+ memory T cells (TCM) in the peripheral
blood was higher in Group T than that in Group C 10 weeks after HIV DNA vaccine
immunization.
IL-15 induced stronger humoral immune response in rhesus monkeys
As shown in Table 1, the titers of anti-env antibody of Group T were higher then those of
Group C 1, 4, and 8 weeks after the rTV boosting, and it was statistically significant 1 week
after the rTV boosting (p<0.05), indicating plasmid IL-15 could enhance the long-term
humoral immune response in rhesus monkeys.
Discussion
HIV vaccines remain to be a major area for investigators in immunology, virology, and
clinical medicine since HIV was identified in the last century. However, the researches of
HIV haven’t made clinically significant progress until one clinical trial carried out by USA
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and Thailand indicated that the ALVAC-HIV and AIDSVAX B/E vaccine regimen may
reduce the risk of HIV infection, although only marginally (13).
IL-15 plays an important role in generation and maintenance of memory CD8+ T cells
(8,14-17), so it is a good candidate for the enhancement of cellular immune response. In this
study, the percentages of effector memory CD8+ T cells of Group T were higher than those
of Group C in the time points we tested, which indicates that IL-15 promotes effector T cells
into memory T cells. When exposed to the same antigen again, specific effector memory
CD8+ T cells can respond to the antigen and convert to CTL rapidly, so it is helpful for long-
term immune function of the vaccine. Also, the percentage of central memory CD8+ T cells
was increased 10 weeks after the DNA immunization. The mechanism might be that the
effector memory CD8+ T cells induced by IL-15 were converted to central memory CD8+ T
cells.
It has been recently reported that high doses of IL-15 decrease the production of IFN-γ and
the proliferation of CD4+ and CD8+ T cells and TCM levels in the proliferating CD4+ and
CD8+ T cells in an influenza non-human primate model (18). Therefore, it may be related to
the dose of IL-15 plasmid as IL-15 did not significantly enhance the HIV-specific CD8+
cellular immune response in our study. Test with larger sample size and dose range should
be carried out before a definite conclusion can be reached.
Our study suggest that IL-15 could increase the percentage of effector memory CD8+ T cells
in rhesus macaque peripheral blood, and enhance the long-term immune response of vaccine
to some extent. Meanwhile, a recent report demonstrated in non-human primates that IL-15
administration expands memory CD8+ and CD4+ T cells, and natural killer (NK) cells in the
peripheral blood, with minimal increases in CD4+CD25+Foxp3+ regulatory T cells, and
intermittent administration of IL-15 has been safe for more than 3 weeks (19). So IL-15
would be an effective adjuvant for HIV vaccine with further optimization of the dose and
dosing schedule.
Acknowledgments
This work is sponsored by grant (2006CB504205) from the National Program for Key Basic Research Project,
Ministry of Science and Technology, P.R. China, and grant from NIH CIPRA (1U19AI51915-02) for Dr. Wei He.
We are grateful to Dr. Yiming Shao(Division of Virology and Immunology, National Center for AIDS/STD
Control and prevention, Chinese Center for Disease Control and Prevention) for the gift of pDRVISV1.0 plasmid.
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