10124287232590Cell Mol ImmunolCell. Mol. Immunol.Cellular & molecular immunology1672-76812042-022620871629337640710.1038/cmi.2010.44NIHMS220070ArticleIL-15 increases the percentage of effector memory CD8+ T cells in rhesus monkeys immunized with HIV vaccineLiShiruiDepartment of Immunology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and School of Basic Medicine, Peking Union Medical College, Beijing 100005, ChinaQiXiangrongNational Institute for Viral Disease Control and Prevention, China CDC, Beijing 100052, ChinaGaoYingyingNational Institute for Viral Disease Control and Prevention, China CDC, Beijing 100052, ChinaHaoYanlingDivision of Virology and Immunology, National Center for AIDS/STD Control and prevention, Chinese Center for Disease Control and Prevention, Beijing 100050, ChinaCuiLianxianDepartment 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, ChinaRuanLiNational Institute for Viral Disease Control and Prevention, China CDC, Beijing 100052, ChinaHeWeiDepartment 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, ChinaCorresponding 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.cn15720102792010112010186201276491494

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.

adjuvanteffector memory CD8+ T cellHIV vaccineIL-15National Institute of Allergy and Infectious Diseases Extramural Activities : NIAIDU19 AI051915-05 || AI
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 MethodsVaccines

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 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.

ResultsIL-15 increased the percentage of effector memory CD8<sup>+</sup> 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 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.

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.

BorrowPLewickiHHahnBHShawGMOldstoneMBVirus-specific CD8+ cytotoxic T-lymphocyte activity associated with control of viremia in primary human immunodeficiency virus type 1 infectionJ Virol199468610361108057491WilsonJDOggGSAllenRLDavisCShaunakSDownieJDirect visualization of HIV-1-specific cytotoxic T lymphocytes during primary infectionAIDS20001422523310716497SafritJTAndrewsCAZhuTHoDDKoupRACharacterization of human immunodeficiency virus type 1-specific cytotoxic T lymphocyte clones isolated during acute seroconversion: recognition of autologous virus sequences within a conserved immunodominant epitopeJ Exp Med19941794634728294860BarouchDHSantraSSteenbekeTDZhengXXPerryHCDaviesMEAugmentation and suppression of immune responses to an HIV-1 DNA vaccine by plasmid cytokine/Ig administrationJ Immunol1998161187518829712056BolestaEKowalczykAWierzbickiAEppolitoCKanekoYTakiguchiMIncreased level and longevity of protective immune responses induced by DNA vaccine expressing the HIV-1 Env glycoprotein when combined with IL-21 and IL-15 gene deliveryJ Immunol200617717719116785513HalwaniRBoyerJDYassine-DiabBHaddadEKRobinsonTMKumarSTherapeutic vaccination with simian immunodeficiency virus (SIV)-DNA + IL-12 or IL-15 induces distinct CD8 memory subsets in SIV-infected macaquesJ Immunol20081807969797918523260OhSBerzofskyJABurkeDSWaldmannTAPereraLPCoadministration of HIV vaccine vectors with vaccinia viruses expressing IL-15 but not IL-2 induces long-lasting cellular immunityProc Natl Acad Sci U S A20031003392339712626740MaAKokaRBurkettPDiverse Functions of IL-2, IL-15, and IL-7 in Lymphoid HomeostasisAnnu Rev Immunol20062465767916551262CarsonWCaligiuriMAInterleukin-15 as a potential regulator of the innate immune responseBraz J Med Biol Res1998311199686173KutzlerMARobinsonTMChattergoonMAChooDKChooAYChoePYCoimmunization with an optimized IL-15 plasmid results in enhanced function and longevity of CD8 T cells that are partially independent of CD4 T cell helpJ Immunol200517511212315972637LiWLiSHuYTangBCuiLHeWEfficient augmentation of a long-lasting immune responses in HIV-1 gag DNA vaccination by IL-15 plasmid boostingVaccine2008263282329018472194KoestersSAAlimontiJBWachihiCMatuLAnzalaOKimaniJEmbreeJEPlummerFAFowkeKRIL-7Ra expression on CD4+ T lymphocytes decreases with HIV disease progression and inversely correlates with immune activationEur J Immunol20063633634416421946Rerks-NgarmSPitisuttithumPNitayaphanSKaewkungwalJChiuJParisRVaccination with ALVAC and AIDSVAX to Prevent HIV-1 Infection in ThailandNew Engl J Med20093612209222019843557SchlunsKSLefrancoisLCytokine control of memory T-cell development and survivalNat Rev Immunol2003326927912669018OhSPereraLPBurkeDSWaldmannTABerzofskyJAIL-15/IL-15Ralpha-mediated avidity maturation of memory CD8+ T cellsProc Natl Acad Sci U S A2004101151541515915477598SurhCDSprentJHomeostasis of naive and memory T cellsImmunity20082984886219100699KimHRHwangKAParkSHKangIIL-7 and IL-15: biology and roles in T-Cell immunity in health and diseaseCrit Rev Immunol20082832533919166383YinJDaiALaddyDJYanJArangoTKhanASHigh dose of plasmid IL-15 inhibits immune responses in an influenza non-human primates immunogenicity modelVirology2009393495519683780BergerCBergerMHackmanRCGoughMElliottCJensenMCSafety and immunologic effects of IL-15 dministration in nonhuman primatesBlood20091142417242619605850

The immunization protocol of rhesus monkeys. 2mg/ml of HIV DNA and IL-15 plasmid were given intramuscularly at the volume of 0.5ml/plasmid, and 108pfu/ml rTV was given subcutaneously at the volume of 50μl. The animals were divided into 2 groups: Group C was primed with HIV DNA and boosted with rTV, and Group T primed with HIV DNA and IL-15 plasmid and boosted with rTV. The peripheral blood was collected at 10, 18, 22, 23, 26 and 30 weeks after the first immunization.

Increased percentage of CD8+ TEM in CD8+ T cells in rhesus monkey peripheral blood. A The percentages of CD8+ TEM in CD8+ T cells in rhesus monkey peripheral blood. B The percentages of CD8+ TCM in CD8+ T cells in rhesus monkey peripheral blood. The CD8 + /CD45RA-/CCR7 - cells represent effector CD8 + memory T cells (TEM), and CD8+/CD45RA-/CCR7+ cells represent central CD8+ memory T cells (TCM) (12). * p<0.05

The titer of anti-env antibody after the rTV boosting

Group CGroup T
C1C2C3C4T1T2T3T4
1 week after rTV boosting1:161:321:321:321:41:5121:5121:16
4 week after rTV boosting1:161:41:81:3201:641:641:32
8 week after rTV boosting1:41:201:401:81:81:4