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Originally published In Press as doi:10.1074/jbc.M309375200 on October 30, 2003

J. Biol. Chem., Vol. 279, Issue 3, 1720-1728, January 16, 2004
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High Attenuation and Immunogenicity of a Simian Immunodeficiency Virus Expressing a Proteolysis-resistant Inhibitor of NF-{kappa}B*

Ileana Quinto{ddagger}§, Antimina Puca§||, Jack Greenhouse**, Peter Silvera**, Jake Yalley-Ogunro**, Mark G. Lewis**, Camillo Palmieri{ddagger}||, Francesca Trimboli{ddagger}, Russ Byrum{ddagger}{ddagger}, Joseph Adelsberger§§, David Venzon¶¶, Xueni Chen§, and Giuseppe Scala{ddagger}§

From the {ddagger}Department of Clinical and Experimental Medicine, Medical School, University of Catanzaro, 88100 Catanzaro, Italy, the §Department of Biochemistry and Biomedical Technology, Medical School, University "Federico II," 80131 Naples, Italy, the **Southern Research Institute, Frederick, Maryland 21701, {ddagger}{ddagger}Bioqual, Rockville, Maryland 20850, §§Science Applications International Corporation-Frederick Inc., National Cancer Institute Frederick, Maryland 21702, and the ¶¶Biostatistics and Data Management Section, NCI, National Institutes of Health, Bethesda, Maryland 20892

Received for publication, August 25, 2003 , and in revised form, October 24, 2003.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
NF-{kappa}B/I{kappa}B proteins play a major role in the transcriptional regulation of human immunodeficiency virus, type-1 (HIV-1). In the case of simian immunodeficiency virus (SIV) the cellular factors required for the viral transcriptional activation and replication in vivo remain undefined. Here, we demonstrate that the p50/p65 NF-{kappa}B transcription factors enhanced the Tat-mediated transcriptional activation of SIVmac239. In addition, I{kappa}B-{alpha}S32/36A, a proteolysis-resistant inhibitor of NF-{kappa}B, strongly inhibited the Tat-mediated transactivation of SIVmac239. Based on this evidence, we have generated a self-regulatory virus by endowing the genome of SIV-mac239 with I{kappa}B-{alpha}S32/36A; the resulting virus, SIVI{kappa}B-{alpha}S32/36A, was nef-deleted and expressed the NF-{kappa}B inhibitor. We show that SIVI{kappa}B-{alpha}S32/36A was highly and stably attenuated both in cell cultures and in vivo in rhesus macaque as compared with a nef-deleted control virus. Moreover, the high attenuation was associated with a robust immune response as measured by SIV-specific antibody production, tetramer, and intracellular IFN-{gamma} staining of SIV gag-specific T cells. These results underscore the crucial role of NF-{kappa}B/I{kappa}B proteins in the regulation of SIV replication both in cell cultures and in monkeys. Thus, inhibitors of NF-{kappa}B could efficiently counteract the SIV/HIV replication in vivo and may assist in developing novel approaches for AIDS vaccine and therapy.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Live attenuated simian immunodeficiency virus (SIV)1 strains deleted of the nef region have provided a sterilizing immunity against homologous and heterologous virus challenge in rhesus macaque (1–5). Unfortunately, the continuous and error-prone replication of the nef-deleted virus gave rise to pathogenic escape mutants (6–9). To improve safety, multiply deleted SIV strains lacking the accessory genes nef, vpr, vpx, vif (10), the regulatory rev and tat genes (11, 12), or the leader RNA sequences (13) were generated with higher levels of attenuation. However, viral attenuation in vivo correlated inversely with the degree of the SIV-specific antibody response and protection from the subsequent viral challenge (14). Attenuation was also achieved by deleting N-linked glycosylation sites in gp120 envelope protein (15, 16), suggesting a more efficient eradication of deglycosylated viruses by antibody- and T cell-mediated immune response. Nevertheless, reversion of glycosylation site mutants was observed (15), indicating that viral replication still occurred despite the efficient immune control of deglycosylated SIV. Here, we describe a gain-of-function strategy to generate a highly attenuated SIV strain endowed with a transcriptional repressor for studies in primate models. SIV and HIV-1 share a similar genome organization and modality of infection. In particular, the LTR of both retroviruses contain a TATA box, Sp1, and NF-{kappa}B cis sequences (17) and transcribe for a 5'-untranslated region that generates the RNA stem loop binding to Tat, the viral elongation factor (18, 19). NF-{kappa}B plays a major role in the transcriptional activation and replication of HIV-1 (20). In fact, NF-{kappa}B proteins activate the expression of HIV-1 by binding to NF-{kappa}B sites of LTR (21, 22) and synergize with Tat-mediated transactivation (23). Moreover, the deletion of the NF-{kappa}B sites in the LTR (24) as well as the expression of NF-{kappa}B inhibitors (25, 26) strongly impair HIV-1 replication. In the case of SIVmac239, the NF-{kappa}B-Sp1 core enhancer in the LTR was described to be either dispensable (27) or required (28, 29) for efficient viral transcription and growth. In this study, we report that I{kappa}B-{alpha}S32/36A, a proteolysis-resistant inhibitor of NF-{kappa}B (30), potently represses the expression and replication of SIVmac239 and can be inserted into the viral genome in order to generate an in vivo highly attenuated and immunogenic virus.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Plasmids and Gene Expression Assays—To generate pSIVLTRluc, the sequence of SIVmac239 (GenBankTM accession number M33262 [GenBank] ) from nucleotides 257–950 was amplified by PCR from the plasmid p239SpE3' (NIH AIDS Research & Reference Reagent Program) with the forward primer 5'-GCTCTAGATGGAAGGGATTTATTACAG-3' and the reverse primer 5'-CCGCTCGAGTACTTCTAAAATGGCAGCT-3'. The PCR product was digested with XbaI and XhoI and ligated to NheI/XhoI-digested pGL3-basic (Promega, Leiden, The Netherlands). The construct was verified by DNA sequencing. The plasmids pRc/CMV, pCMVp50, pCMVp65, pCMV-HA-I{kappa}B-{alpha}S32/36A, pNLI{kappa}B-{alpha}S32/36A and pNLI{kappa}B-antisense were as previously described (26). pYC5 expressing the tat gene of HIV-2 was kindly provided by K.-T. Jeang. pMA239 expressing the SIVmac239 genome was a gift from T. Yilma. For in transient gene expression assays, COS-1 cells (3 x 105) were transfected with pSIVLTRluc (1 µg) in presence or absence of pYC5 (0.5 µg), and the indicated plasmids (2 µg) using the calcium phosphate method as previously reported (26). After 48 h, whole cell extracts were analyzed for luciferase activity. The transfection efficiency was measured by co-transfection of pSV-{beta}-galactosidase control vector (1 µg) followed by {beta}-galactosidase assay, as previously described (26). Similarly, COS-1 cells were transfected with pMA239 (1 µg) in presence of pNLI{kappa}B-{alpha}S32/36A (5 µg), pNLI{kappa}B-antisense (5 µg), or pRc/CMV (5 µg). The production of SIV virions was measured by p27 ELISA assay in culture supernatants at 48 and 72 h post-transfection using the SIV core antigen kit (Coulter Corp., Hialeah, FL).

Construction of pSIVI{kappa}B-{alpha}S32/36A—The sequence of SIVmac239 from 8997 to 9499 nucleotides was amplified by PCR from p239SpE3' (NIH AIDS Research & Reference Reagent Program) with the primers SIV11, 5'-CCCAAGCTTGCTAGCTAAGTTAAGGCAGG-3' and SIV12, 5'-TTCCGCGGCCGCTATGGCCGACGTCGACTACTCACAAGAGAGTGAGCTC-3'. The PCR product was ligated to pSP73 (Promega) after digestion with Hind III and SalI to generate pSP73SIV11/12. Then, the sequence of SIVmac239 from nucleotides 9682 to 10535 was amplified from p239SpE3' with the primers SIV13, 5'-ACGCGTCGACGCGGCCGCTCTAGACATGTCTCATTTTATAAAAGAA AAG-3' and SIV14, 5'-GGAATTCTAATGTTGGTGGAAACTG-3' and ligated to pSP73SIV11/12 after digestion with SalI and EcoRI to generate the shuttle SIV{Delta}nef. The resulting nef sequence was deleted from nucleotides 9501 to 9681 and replaced with a sequence containing a stop codon after the 56th amino acid of Nef followed by the unique cloning sites SalI and XbaI. The I{kappa}B-{alpha}S32/36A cDNA fused to the FLAG epitope was cloned in sense or antisense orientation in the shuttle SIV{Delta}nef digested with SalI and XbaI to generate the shuttles SIVI{kappa}B-{alpha}S32/36A and SIVI{kappa}B-antisense, respectively. For cloning in sense orientation, I{kappa}B-{alpha}S32/36A was amplified from pCMV-HA-I{kappa}B-{alpha}S32/36A with the primers 5'I{kappa}B-{alpha}, 5'-ACGCGTCGACATGTTCCAGGCGGCCGAGCG-3' and 3'I{kappa}B-{alpha}, 5'-GCTCTAGATCACTTGTCGTCATCGTCTTTGTAGTCTAACGTCAGACGCTGG-3'. For cloning in antisense orientation, I{kappa}B-{alpha}S32/36A was amplified with the primers 5'I{kappa}B-AS, 5'-GCTCTAGAATGTTCCAGGCGGCCGAGCGC-3' and 3'I{kappa}B-AS, 5'-ACGCGTCGACTAGTAACGTCAGACGCTGGCCTCCAAA-3'. Following NheI and EcoRI digestion, the 1.3 kb of the shuttle SIV{Delta}nef or the 2.3-kb fragments of the shuttles SIVI{kappa}B-{alpha}S32/36A and SIVI{kappa}B-antisense were ligated to the 14-kb NheI/EcoR I fragment of pMA239 to generate pSIV{Delta}nef, pSIVI{kappa}B-{alpha}S32/36A, and pSIVI{kappa}B-antisense, respectively. The correct coding frame of the insert was verified by DNA sequencing. The expression of viral proteins was analyzed by transfection of 293 T cells with the viral plasmids (5 µg) and 48 h later by Western blotting of cell extracts (10 µg) with anti-SIVmac251 serum (NIH AIDS Research & Reference Reagent Program) (26). The I{kappa}B-{alpha}S32/36A-FLAG protein was immunoprecipitated from cell extracts (500 µg) using anti-FLAG M2 mAb (Sigma) and detected by Western blotting with I{kappa}B-{alpha} (C-15)-G goat polyclonal IgG (Santa Cruz Biotechnology, Inc., Santa Cruz, CA).

Viral Stocks and Cell Culture Infections—Viral stocks were produced by transfecting 293 T cells with viral plasmids and evaluated by RT assay and p27gag antigen using the SIV core antigen kit (Coulter Corp., Hialeah, FL) as previously reported (26). For cell culture infections, CEMx174 (105 cells) and rhesus macaque PBMC (2 x 105 cells) stimulated with concanavalin A were infected with 105 and 2 x 105 cpm RT activity of viral stocks, respectively. Viral growth was monitored by RT activity, as previously described (26). For in vitro passages, CEMx174 (105 cells) were infected with 105 cpm RT activity. At the RT peak of each infection, virus was collected and used for infections in the next passage. For analysis of nef region, viruses were collected at the RT peak of each passage and the viral RNA was analyzed by RT-PCR using the primers SIV15, 5'-TCTGCGACAGAGACT-3' (9377–9391 nucleotides of SIVmac239) and SIV16, 5'-TCCTGCCAATCTGGT-3' (9791–9805 nucleotides of SIVmac239). Titers of viral stocks for monkey infection were measured as TCID50 by using CEMx174 cells.

Animal Infection Studies—Mamu-A*01-positive juvenile rhesus macaques were maintained in accordance with the American Association for Accreditation of Laboratory Animal Care Standards and housed in a biosafety level 2 facility. Viral infection was performed by intravenous inoculation of 100 TCID50 of viral stocks. The infected monkeys were monitored for plasma viral load by detecting SIV RNA using a real-time reverse transcription-PCR with a threshold sensitivity of 200 viral RNA copies Eq/ml (31). The gag region was amplified by using the following primers and probe: SIV-F, 5'-AGTATGGGCAGCAAATGAAT-3'; SIV-R, 5'-TTCTCTTCTGCGTGAATGC-3'; SIV-P, 6FAM-AGATTTGGATTAGCAGAAAGCCTGTTGGA-TAMRA. For cocultures, PBMC and lymph node cells (106 up to 107) from infected animals were stimulated with 10 µg/ml PHA (Sigma-Aldrich) for 2 days and cultured with PHA-activated PBMC from uninfected monkeys or with CEMx174 cells. Cultures were monitored by RT activity and p27 production over 5 weeks of coculture.

To analyze the nef region, viral RNA was reverse-transcribed and amplified using Superscript one-step RT-PCR for long template (Invitrogen Life Technologies) with the primers SIV15 and SIV16 above described. The PCR product (1 µl) was re-amplified with the primers SIV 17, 5'-GCGTGGGGAGACTTAT-3' (9401–9416 nucleotides of SIV-mac239) and SIV 18,5'-CTTGCACTGTAATAAATCCC-3' (9723–9742 nucleotides of SIVmac239). The PCR product was analyzed by liquid hybridization with a {gamma}-32P-labeled I{kappa}B-{alpha} probe followed by electrophoresis on 10% acrylamide gel and autoradiography. The I{kappa}B-{alpha} probe was: 5'-TCCTGACCTGGTGTCACTC-3'. For gag analysis, viral RNA was amplified by RT-PCR with primers SIV-F and SIV-R above described, and the PCR products were detected by liquid hybridization with {gamma}-32P-labeled SIV-P probe.

Genomic DNA was extracted from lymph node cells using the Wizard Genomic DNA purification kit (Promega). The nef region was amplified by PCR of genomic DNA (100 ng) using the primers SIV25, 5'-TCCTCAGGACTGAACTGACC-3' (9303–9322 nucleotides of SIVmac239) and SIV22, 5'-TACATCAAGAAAGTGGGCGTTC-3' (10185–10206 nucleotides of SIVmac239) followed by nested PCR with the primers SIV17 and SIV18. The PCR products were analyzed by liquid hybridization with {gamma}-32P-labeled I{kappa}B-{alpha} probe. Rhesus macaques were screened for the presence of the MAMU-A*01 allele by PCR of genomic DNA using the primers MAMU-A*01F, 5'-GACAGCGACGCCGCGAGCCAA-3' and MAMU-A*01R, 5'-GCTGCAGCGTCTCCTTCCCC-3' as described (32).

Immune Response—Antibody immune response was analyzed by ELISA using HIV-2 microplate EIA (Bio-Rad Laboratories, Hercules, CA). Plasma samples were tested at serial dilutions. The identification of SIV Gag p11C-specific T cells was performed by tetramer staining of freshly isolated PBMC (33). Briefly, PBMC (106 cells) were stained with phycoerythrin-labeled tetrameric Mamu-A*01/SIV Gag p11C epitope (CTPYDINQM) (NIH, MHC Tetramer Core Facility) for 15 min at room temperature followed by staining with anti-human CD8 monoclonal antibody conjugated with peridinin chlorophyl protein (BD Biosciences, San Diego, CA) for further 15 min. Tetramer+-CD8+ T cells were measured by using a FACScalibur flow cytometer and CellQuest software (BD Biosciences). To detect IFN-{gamma}+ CTL, PBMC were stimulated for 2 h with a 20-mer SIVmac239 Gag peptide (2.5 µg/ml) spanning the amino acids 181–200 of SIVmac239 Gag (CTPYDINQMLNCVGDHQAAM) (NIH AIDS Research and Reference Reagent Program) in 5% fetal bovine serum RPMI 1640. As controls, PBMC were stimulated with staphylococcus-enterotoxin B or left unstimulated. After overnight incubation with (1 µg/ml) of Brefeldin A (BD PharMingen, San Diego, CA), PBMC were stained with peridinin chlorophyl protein-labeled anti-human CD8 monoclonal antibody (BD Biosciences). Then, cells were fixed and permeabilized using Cytofix/Perm kit (BD PharMingen) followed by staining with phycoerythrin-labeled anti-human IFN-{gamma} (BD PharMingen). IFN-{gamma}+-CD8+ T cells were measured by flow cytometry. Levels of peripheral CD4- and CD8-positive T cells were determined by flow cytometry by using the following antibodies: CD3-FITC (BD Pharmingen), CD4-PE and CD8-PE (BD Biosciences).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Inhibition of SIVmac239 Expression by I{kappa}B-{alpha}S32/36A—We first analyzed the LTR-driven transcription of SIVmac239 in response to I{kappa}B-{alpha}S32/36A. COS-1 cells were transfected with pSIVLTRluc and a Tat-expressing plasmid in the presence or absence of pCMV-HA-I{kappa}B-{alpha}S32/36A (Fig. 1a). As measured by luciferase activity, I{kappa}B-{alpha}S32/36A strongly inhibited the Tat-mediated transactivation of SIV-LTR. The inhibition by I{kappa}B-{alpha} S32/36A was likely exerted by squelching NF-{kappa}B proteins required for full activation of SIV-LTR. In fact, co-transfection of p50 and p65 subunits of NF-{kappa}B enhanced the Tat-mediated transactivation of SIV-LTR (Fig. 1b). We also tested the effect of I{kappa}B-{alpha}S32/36A on SIV production by co-transfecting the SIV-mac239 genome alone or together with HIV-1 plasmids expressing I{kappa}B-{alpha}S32/36A in sense (pNLI{kappa}B-{alpha}S32/36A) or antisense (pNLI{kappa}B-antisense) orientation (26) (Fig. 1c). I{kappa}B-{alpha}S32/36A exerted a strong inhibition of SIV production as measured by p27 Gag detection in culture supernatants. These results indicate that the Tat-mediated expression of SIV is enhanced by NF-{kappa}B and is inhibited by I{kappa}B-{alpha}S32/36A.



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  		FIG. 1.
I{kappa}B-{alpha} S32/36A inhibits the expression of SIVmac239 and the production of SIV virions. a, expression of pSIVLTRluc in COS-1 cells in the presence or absence of Tat and pCMV-HA-I{kappa}B-{alpha}S32/36A or pRc/CMV empty plasmid. Luciferase activity is expressed as arbitrary units/100 µg of proteins (mean ± S.E.) of five independent experiments. b, expression of pSIVLTRluc in COS-1 cells in the presence or absence of Tat and pCMVp50, pCMVp65, or pRc/CMV empty plasmid. Luciferase activity is expressed as arbitrary units/100 µg of proteins (mean ± S.E.) of four independent experiments. c, production of SIVmac239 virions in COS-1 after transfection of pMA239 in presence of an empty plasmid (pRc/CMV), or plasmids that express the HIV-1 genome endowed with I{kappa}B-{alpha}S32/36A in sense (pNLI{kappa}B-{alpha}S32/36A) or antisense orientation (pNLI{kappa}B-antisense). Results are expressed as viral p27 (ng/ml, mean ± S.E.) of four independent experiments.

 
Attenuation of SIVI{kappa}B-{alpha} S32/36A in Cell Cultures—Next, we generated a SIVmac239 expressing I{kappa}B-{alpha}S32/36A, SIVI{kappa}B-{alpha}S32/36A, by inserting the I{kappa}B-{alpha}S32/36A cDNA fused to the FLAG epitope into the nef region of SIVmac239 (Fig. 2a). As a control, we generated a SIVmac239 carrying the I{kappa}B-{alpha}S32/36A insert in antisense orientation (Fig. 2a). In both recombinant viruses, we deleted the nef coding region from nucleotides 9501 to 9681 of SIVmac239 leaving in place the sequence that overlaps with the env gene and codes for the first 56 amino acids of Nef (Fig. 2a). We detected the expression of viral proteins and I{kappa}B-{alpha}S32/36A by Western blotting of cell extracts upon transfection of 293 T cells with the viral plasmids (Fig. 2, b and c). As expected, I{kappa}B-{alpha}S32/36A was produced exclusively by SIVI{kappa}B-{alpha}S32/36A (Fig. 2c).



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  		FIG. 2.
Construction of SIVmac239 expressing I{kappa}B-{alpha}S32/36A. a, schematic representation of recombinant SIVmac239 genome carrying the I{kappa}B{alpha}S32/36A cDNA integrated in sense or antisense orientation into nef. b, expression of viral proteins in 293 T cells transfected with the indicated viral plasmids. Immunoblot analysis was performed with anti-SIVmac251 serum. c, expression of I{kappa}B-{alpha}S32/36A in 293 T cells transfected with the indicated viral plasmids. I{kappa}B-{alpha}S32/36A was immunoprecipitated with anti-FLAG M2 mAb, and the immunoblot analysis was performed with I{kappa}B-{alpha} (C-15) goat polyclonal IgG.

 
Next, we analyzed the viral growth in CEMx174 cells and in rhesus macaque PBMC (Fig. 3a). SIVI{kappa}B-{alpha}S32/36A was highly attenuated as compared with the wild-type SIVmac239, the nef-deleted virus (SIV{Delta}nef), and the SIVI{kappa}B-antisense (Fig. 3a, left panel). An even stronger attenuation was observed in PBMC where SIVI{kappa}B-{alpha}S32/36A was unable to grow (Fig. 3a, right panel). SIVI{kappa}B-{alpha}S32/36A maintained the attenuated phenotype (Fig. 3b) as well as the I{kappa}B-{alpha}S32/36A insert (Fig. 3c) in eight serial passages in CEMx174 cultures. In contrast, the insert was rapidly rearranged and lost in SIVI{kappa}B-antisense (Fig. 3c).



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  		FIG. 3.
Growth of SIVI{kappa}B-{alpha}S32/36A in cell culture. a, SIVI{kappa}B-{alpha}S32/36A is attenuated in CEMx174 (left panel) and in rhesus macaque PBMC (right panel). Cells were infected with viral stocks as detailed under "Experimental Procedures," virus production was monitored by RT activity of culture supernatants. The results are representative of four independent experiments. b, SIVI{kappa}B-{alpha}S32/36A maintains the attenuated phenotype over eight serial passages in CEMx174. For each passage, virus was collected at the peak of infection and used to infect CEMx174 in the following passage as detailed under "Experimental Procedures." c, SIVI{kappa}B-{alpha}S32/36A maintains the I{kappa}B-{alpha} insert in eight serial passages in CEMx174. Viral RNA was extracted from virions collected at the peak of infection of each passage; the nef region amplified by RT-PCR with primers SIV15 and SIV16 is 1233 bp in SIVI{kappa}B-{alpha}S32/36A and SIVI{kappa}B-antisense, 428 bp in SIVmac239 and 263 bp in SIV{Delta}nef. Marker of molecular weights is a 100-bp DNA ladder.

 
In Vivo Attenuation of SIVI{kappa}B-{alpha}S32/36A—To analyze the in vivo infectivity of SIVI{kappa}B-{alpha}S32/36A, we intravenously injected rhesus macaques with 100 TCID50 of SIVI{kappa}B-{alpha}S32/36A (529, 904, 540, and 893) or SIVI{kappa}B-antisense (WBA, 374, 825, and 599). In the period of acute infection extending from week 0 through week 8, the group of animals infected with SIVI{kappa}B-{alpha}S32/36A had peaks of viremia lower than animals infected with control antisense virus (p = 0.029 by the Wilcoxon ranksum test, Fig. 4). From week 8 to 16, the group infected with SIVI{kappa}B-{alpha}S32/36A all had undetectable virus showing an overall significant difference with the control group infected with antisense virus (p = 0.028 by the Wei-Johnson method). After that time, the animals infected with the sense virus were aviremic for up to 2 years post-infection with a transient viremia in monkey 529 observed at weeks 17 and 18, while animals infected with control antisense virus were persistently viremic with the exception of monkey WBA (Fig. 4). Accordingly, no virus production was detected in cocultures of PBMC from SIVI{kappa}B-{alpha}S32/36A-infected animals (529, 904, 540, 893) at months 3, 6, 12, and 24 post-infection (Table I). In parallel, cocultures of PBMC from the control SIVI{kappa}B-antisense-infected monkeys produced virus at months 3, 6, 12, and 24 in animals 374, 825, and 599, and at month 3 in animal WBA (Table I). Moreover, cells obtained from two inguinal lymph nodes at 1 year post-infection produced virus only in the case of animals infected with the control antisense construct (WBA, 374, 825, 599; Table I). At 2 years post-infection, CD4+ T cells counts were stably maintained in the animals infected with SIVI{kappa}B-{alpha}S32/36A while they were substantially reduced in two animals of the control group (WBA, 374; Fig. 4). Altogether, these data indicate that the expression of I{kappa}B-{alpha}S32/36A coupled with nef deletion conferred a stronger attenuation of SIV in vivo as compared with an SIV nef-deleted control (SIVI{kappa}B-antisense).



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  		FIG. 4.
Attenuation of SIVI{kappa}B-{alpha}S32/36A in rhesus macaques. Animals were intravenously injected with 100 TCID50 of SIVI{kappa}B-antisense or SIVI{kappa}B-{alpha}S32/36A. The plasma viremia (left panels) and CD4+ T cell counts (right panels) were measured in rhesus macaques infected with SIVI{kappa}B-antisense or SIVI{kappa}B-{alpha}S32/36A as detailed under "Experimental Procedures."

 


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  		TABLE I
Virus production in PBMC and lymph nodes of infected monkeys

PBMC or lymph node cells (106 up to 107) obtained from infected animals were cultured (1:1 ratio) with PHA-activated PBMC from uninfected monkeys for five weeks. Culture supernatants were analyzed for viral production by measuring RT activity and p27 production.

 
Next, we analyzed the stability of the I{kappa}B-{alpha} insert in plasma virions and lymph node cells. At week 3 of primary infection, the I{kappa}B-{alpha}S32/36A insert was completely (WBA, 374) or partially (825, 599) lost when placed in antisense orientation (Fig. 5, lanes 1–4), while it was maintained in the viral genome when placed in sense orientation (529, 904, 540, 893) (Fig. 5, lanes 12–15). At week 18 post-infection, virions produced in the antisense-infected animals had completely lost the I{kappa}B-{alpha}S32/36A antisense insert with the exception of 599 (Fig. 5, lanes 6–9). At the same time, animals 904, 540, and 893 infected with the sense construct tested negative in this assay (Fig. 5, lanes 18–20) consistently with the lack of detectable plasma viremia shown in Fig. 4; virions produced in 529 were depleted of the I{kappa}B-{alpha} insert (Fig. 5, lane 17). These findings indicate that viral attenuation correlated with the maintenance of I{kappa}B-{alpha}S32/36A expressed in sense orientation; in fact, the lack of the sense I{kappa}B-{alpha}S32/36A insert was associated with viremia in 529, while the maintenance of the antisense I{kappa}B-{alpha}S32/36A insert did not provide attenuation in 599. At 1 year post-infection, the genomic DNA from two inguinal lymph nodes of each monkey was analyzed for virus integration. In the animals infected with SIVI{kappa}B-antisense, the nef region was largely deleted (Fig. 6, upper panel, lanes 1–4). In animals infected with SIVI{kappa}B-{alpha}S32/36A, the nef region was amplified as a band of the expected size; smaller bands were also detected in 529, 904, and 540 (Fig. 6, upper panel, lanes 5–8). By liquid hybridization, the I{kappa}B-{alpha}S32/36A insert was present in the nef-amplified region of animals infected with SIVI{kappa}B-{alpha}S32/36A (Fig. 6, lower panel, lanes 5–8), while it was absent in animals infected with the control antisense virus with the exception of 599 (Fig. 6, lower panel, lanes 1–4). These findings are consistent with the higher genomic stability of SIVI{kappa}B-{alpha}S32/36A as compared with the SIVI{kappa}B-antisense control virus.



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  		FIG. 5.
Analysis of the I{kappa}B-{alpha} insert in plasma virions. The analysis of nef and gag regions in plasma virions derived from infected Rhesus macaques was performed at weeks 3 and 18 post-infection. The nef region was amplified from viral RNA by RT-PCR using the primers SIV15 and SIV16 followed by nested PCR with primers SIV17 and SIV18. Viral stocks of SIVI{kappa}B-{alpha}S32/36A and SIVI{kappa}B-antisense were analyzed as controls. The expected size of nef amplified product of SIVI{kappa}B-{alpha}S32/36A and SIVI{kappa}B-antisense is 1147 bp (upper panel). The nef PCR products were analyzed by liquid hybridization with the I{kappa}B-{alpha} probe (center panel). The gag region was amplified by RT-PCR with primers SIV-F and SIV-R and detected by liquid hybridization with SIV-P probe (lower panel).

 



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  		FIG. 6.
Analysis of virus integration in lymph node cells. The nef region was amplified from genomic DNA of lymph node cells with the primers SIV22 and SIV25 followed by nested PCR with primers SIV17 and SIV18. The viral plasmids pSIVI{kappa}B-{alpha}S32/36A and pSIVI{kappa}B-antisense were amplified as controls. The nef PCR product is 1147 bp in SIVI{kappa}B-{alpha}S32/36A and SIVI{kappa}B-antisense (upper panel). The nef product was analyzed by liquid hybridization with the I{kappa}B-{alpha} probe (lower panel).

 
Analysis of Immune Response—Next, we analyzed the immune response elicited in infected monkeys. SIV-specific antibodies were detected at week 2 post-infection in all the animals and increased to higher levels throughout 2 years of post-infection observation (Fig. 7a). Antibody production did not correlate with the ability to control the viremia since the group of animals infected with antisense construct showed similar or even higher titers of SIV-specific antibodies as compared with the group of animals infected with SIVI{kappa}B-{alpha}S32/36A (Fig. 7a).



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  		FIG. 7.
Immune response post-infection. a, antibody production. Titers of anti-SIV antibodies were determined in plasma of infected animals by ELISA as detailed under "Experimental Procedures." b, tetramer staining. p11C Gag tetramer+-CD8+ T lymphocytes were measured by FACS and expressed as percentages of positive cells. c, intracellular IFN-{gamma} production. IFN-{gamma} +-CD8+ T lymphocytes were measured by FACS and expressed as percentages of positive cells.

 
In further studies, we measured the number of p11C Gag-specific CD8+ T lymphocytes. Over 2 years of observation, the percentages of p11C Gag-specific CD8+ T lymphocytes ranged between 0.1 and 2.15 in the group of animals infected with SIV-I{kappa}B{alpha}S32/36A, and between 0.1 and 0.6 in the group of animals infected with antisense construct (Fig. 7b) with no significant difference between the two groups (p = 0.14 by the Wilcoxon ranksum test). In parallel, we analyzed the frequency of CD8+ T cells producing intracellular IFN-{gamma} in response to the p11C Gag peptide. At 2 months post-infection, a substantial population of IFN-{gamma}+-CD8+ T cells was detected in three animals (904, 540, and 893) of the group infected with the sense construct, and in one animal (WBA) of the group infected with the antisense control construct (Fig. 7c). In the following months, the population of IFN-{gamma}+-CD8+ T cells appeared at variable levels in all animals of both groups. At 24 months post-infection, all animals maintained a substantial number of IFN-{gamma}+-CD8+ T cells with the exception of animal WBA (Fig. 7c). No significant difference was observed in the number of IFN-{gamma}+-CD8+ T cells between the two groups of animals (p = 0.89 by the Wilcoxon ranksum test). Consistent with previous reports in SIV-infected monkeys (34) and HIV-1-infected subjects (35), no statistically significant correlation was observed between the ability to control viremia and the levels of p11C Gag-specific T lymphocytes (p = 0.11 by the Spearman rank test) or IFN-{gamma}+-CD8+ T cells (p = 0.14 by the Spearman rank test).

These results show that SIV-specific antibody production and virus-specific T cell activity were induced in both groups of animals at similar levels, thus indicating that the ability to control viremia in SIVI{kappa}B-{alpha}S32/36A-infected animals was due to the poor growth ability of the virus. Of interest, a long-lasting immune response was maintained in SIVI{kappa}B-{alpha}S32/36A-infected monkeys even in the absence of detectable viremia over 2 years post-infection.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
SIV strains deleted of the nef gene are attenuated in vivo (6) and have been proposed as live attenuated vaccine viruses (1–5); however, they can reconstitute the Nef function as a consequence of rearrangements of the nef region (7–9). The genomic instability of nef-deleted live attenuated viruses has precluded their use as vaccine candidates for AIDS. To overcome these hurdles, we have developed a novel strategy of attenuation by coupling the loss of Nef with the gain of a repressor of viral expression. First, we have shown that p50/p65 NF-{kappa}B complex increased the Tat-mediated transactivation of the SIV LTR (Fig. 1b), while I{kappa}B-{alpha} S32/36A, a proteolysis-resistant inhibitor of NF-{kappa}B, potently inhibited the expression and production of SIV (Fig. 1, a and c). I{kappa}B-{alpha} was previously shown to inhibit the NF-{kappa}B-mediated expression and replication of HIV-1 (25, 26, 30) and to interfere with the Rev function (36). Thus, as in the case of HIV-1 (21–26, 30, 36), NF-{kappa}B/I{kappa}B proteins may play a major role in the regulation of SIV transcription and replication. The role of NF-{kappa}B in the SIV regulation has been controversial due to studies with LTR-deleted SIV strains. In fact, the NF-{kappa}B-Sp1 enhancer in SIV-mac239 LTR was shown to be required (28) and sufficient (29) for viral growth, or dispensable (27). In contrast to previous studies, we have analyzed the effect of NF-{kappa}B/I{kappa}B proteins on SIVmac239 expression without modifying the complex organization of LTR; indeed, deletions of the LTR may create alternative regulatory elements that could be relevant in the context of a mutated virus. The redundancy of NF-{kappa}B and Sp1 sites in the HIV/SIV LTR could ensure the activation of viral expression in different cell types. In this regard, NF-{kappa}B and Sp1 can individually contribute to the HIV-1 promoter activity (37). Moreover, SIV strains that lack the NF-{kappa}B enhancer (27, 38) or the Sp1 enhancer (27) are still able to replicate efficiently. Altogether, these findings indicate that HIV/SIV may redundantly use either NF-{kappa}B or Sp1 for transcription.

To evaluate the inhibition of viral growth by I{kappa}B-{alpha}, we endowed the SIVmac239 genome with I{kappa}B-{alpha}S32/36A in sense or antisense orientation. Only the sense orientation allowed the expression of I{kappa}B-{alpha}S32/36A. We observed that SIV expressing I{kappa}B-{alpha}S32/36A, SIVI{kappa}B-{alpha}S32/36A, was potently attenuated in cell cultures and in monkeys. Differently from the control antisense virus, SIV expressing I{kappa}B-{alpha}S32/36A was unable to grow in PBMC (Fig. 3a), showed 10-fold lower levels of viremia in the course of acute primary infection, and was undetectable during the following 2 years of post-acute infection (Fig. 4). Moreover, SIVI{kappa}B-{alpha}S32/36A-infected monkeys showed conserved levels of peripheral CD4+ T cells with absence of clinical abnormalities over the 2 years of post-infection observation (Fig. 4). Differently, two of the four animals infected with the control SIVI{kappa}B-antisense (WBA and 374) experienced decreasing levels of CD4+ T-cells (Fig. 4). In particular, in animal WBA the decay in CD4+ T cells indicated that the SIVI{kappa}B-antisense replication was persistently ongoing despite the lack of detectable plasma viremia from week 19 post-infection. Accordingly, cells from inguinal lymph nodes produced virus only in the case of SIVI{kappa}B-antisense-infected animals including the monkey WBA with undetectable viremia in late infection (Table I). Thus, in the same genetic background, SIVI{kappa}B-{alpha}S32/36A showed a higher level of attenuation as compared with SIVI{kappa}B-antisense. Since the two viruses differ for the ability to express I{kappa}B-{alpha}S32/36A, the stronger attenuation of SIV I{kappa}B-{alpha}S32/36A was essentially due to the I{kappa}B-{alpha} repressor inhibiting the SIV expression.

The attenuation of SIVI{kappa}B-{alpha}S32/36A correlated with the maintenance of the I{kappa}B-{alpha}S32/36A insert as shown by serial passages in cell cultures (Fig. 3c). This pattern was also observed in monkeys, where SIVI{kappa}B-{alpha}S32/36A kept the I{kappa}B-{alpha}S32/36A insert as observed in plasma virions produced in the course of primary infection (Fig. 5) and in lymph node cells at 1 year post-infection (Fig. 6). At the same time of observation, the control SIVI{kappa}B-antisense rapidly lost the I{kappa}B insert (Figs. 3c, 5, and 6). These results underscore the higher genomic stability of SIVI{kappa}B-{alpha}S32/36A as compared with SIVI{kappa}B-antisense and suggest that I{kappa}B-{alpha}S32/36A exerted anti-mutator activity by reducing the replicative rate of the virus.

In multiply-deleted SIV strains the degree of attenuation correlated inversely with the level of SIV-specific antibodies (14). The lack of crucial viral epitopes in multiply-deleted viruses may have enabled the induction of a broadly protective immune response (14). Differently from previous approaches, the strategy of attenuation by gain-of-repressor decreased the replication rate of SIV while preserving all of the viral proteins except Nef. Indeed, SIVI{kappa}B-{alpha}S32/36A-infected animals elicited a robust antibody and T cell-mediated immune response that was comparable to the control SIVI{kappa}B-antisense (Fig. 7a). The strong immunogenicity of SIVI{kappa}B-{alpha}S32/36A represents a novel attribute of a highly attenuated virus. In fact, a highly attenuated SIVmac239 lacking nef, vpr, vpx, and upstream sequences in U3 showed a pattern of viral attenuation comparable to SIVI{kappa}B-{alpha}S32/36A (10) but was unable to raise an efficient SIV-specific antibody response (14). The effective immune response coupled with the intrinsically slow replication of the SIVI{kappa}B-{alpha}S32/36A may have contributed to the eradication of possible escape mutants lacking the insert, as documented in monkey 529. In fact, monkey 529 that showed an occasional viremia at weeks 17 and 18 as a consequence of a deletion of the I{kappa}B insert, was aviremic during the following 2 years of post-infection observation with PBMC, and lymph node cultures tested negative for viral production (Figs. 4 and 5 and Table I).

In summary, we have demonstrated that I{kappa}B-{alpha}S32/36A is a potent repressor of SIVmac239 replication. Consistent with these results, we have previously reported that an HIV-1 strain expressing I{kappa}B-{alpha}S32/36A showed a strong attenuation in primary cultures of human PBMC (26). Thus, the in vivo studies reported here may have a direct relevance for HIV-1 infection and may assist in developing new strategies of HIV-1 attenuation in vivo. Indeed, our studies underscore the in vivo relevance of NF-{kappa}B/I{kappa}B proteins in the regulation of SIV/HIV replication and indicate that inhibition of NF-{kappa}B signaling interferes with viral progression in vivo.


   FOOTNOTES
 
* This work was supported by grants from Istituto Superiore di Sanità-National Research Program on AIDS, and Ministero dell'Istruzione, dell'Università e della Ricerca. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

|| Recipients of a fellowship from Fondazione Italiana per la Ricerca sul Cancro. Back

To whom correspondence should be addressed. Tel.: 39-081-7463157; Fax: 39-081-7463150; E-mail: quinto{at}dbbm.unina.it.

1 The abbreviations used are: SIV, simian immunodeficiency virus; HIV-1, human immunodeficiency virus, type 1; LTR, long terminal repeats; RT, reverse transcriptase; PBMC, peripheral blood mononuclear cells; TCID50, 50% tissue culture infectious doses; PHA, phytohemagglutinin; IFN-{gamma}, interferon-{gamma}; HA, hemagglutinin; ELISA, enzyme-linked immunosorbent assay; mAb, monoclonal antibody; FACS, fluorescent-activated cell sorter. Back


   ACKNOWLEDGMENTS
 
We thank K. T. Jeang for pYC5 and T. Yilma for pMA239. We also thank G. Franchini for helpful discussions. We are grateful to the National Institutes of Health AIDS Research and Reagent Program for providing plasmids, antibodies, and peptides.



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 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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A. Puca, G. Fiume, C. Palmieri, F. Trimboli, F. Olimpico, G. Scala, and I. Quinto
I{kappa}B-{alpha} Represses the Transcriptional Activity of the HIV-1 Tat Transactivator by Promoting Its Nuclear Export
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