Protecting from r5-tropic hiv: individual and combined effectiveness of a hammerhead ribozyme and a single-chain fv antibody that targets ccr5

ABSTRACT The CCR5 chemokine receptor is important for most clinical strains of HIV to establish infection. Individuals with naturally occurring polymorphisms in the CCR5 gene who have

reduced or absent CCR5 are apparently otherwise healthy, but are resistant to HIV infection. With the goal of reducing CCR5 and protecting CCR5+ cells from R5-tropic HIV, we used Tag-deleted

SV40-derived vectors to deliver several anti-CCR5 transgenes: 2C7, a single-chain Fv (SFv) antibody; VCKA1, a hammerhead ribozyme; and two natural CCR5 ligands, MIP-1α and MIP-1β, modified

to direct these chemokines, and hence their receptor to the endoplasmic reticulum. These transgenes were delivered using recombinant, Tag-deleted SV40-derived vectors to human CCR5+ cell

lines and primary cells: monocyte-derived macrophages and brain microglia. All transgenes except MIP-1α decreased CCR5, as assayed by immunostaining, Northern blotting, and cytofluorimetry

(FACS). Individually, all transgenes except MIP-1α protected from low challenge doses of HIV. At higher dose HIV challenges, protection provided by all transgenes diminished, the SFv and the

ribozyme being most potent. Vectors carrying these two transgenes were used sequentially to deliver combination anti-CCR5 genetic therapy. This approach gave approximately additive

reduction in CCR5, as measured by FACS and protected from higher dose HIV challenges. Reducing cell membrane CCR5 using anti-CCR5 transgenes, alone or in combinations, may therefore provide

a degree of protection from R5-tropic strains of HIV. Access through your institution Buy or subscribe This is a preview of subscription content, access via your institution ACCESS OPTIONS

Access through your institution Subscribe to this journal Receive 6 print issues and online access $259.00 per year only $43.17 per issue Learn more Buy this article * Purchase on

SpringerLink * Instant access to full article PDF Buy now Prices may be subject to local taxes which are calculated during checkout ADDITIONAL ACCESS OPTIONS: * Log in * Learn about

institutional subscriptions * Read our FAQs * Contact customer support SIMILAR CONTENT BEING VIEWED BY OTHERS CCL2 IS REQUIRED FOR INITIATION BUT NOT PERSISTENCE OF HIV INFECTION MEDIATED

NEUROCOGNITIVE DISEASE IN MICE Article Open access 21 April 2023 C-C MOTIF CHEMOKINE RECEPTOR 2 AND 7 SYNERGISTICALLY CONTROL INFLAMMATORY MONOCYTE RECRUITMENT BUT THE INFECTING VIRUS

DICTATES MONOCYTE FUNCTION IN THE BRAIN Article Open access 24 April 2024 ADENO-ASSOCIATED VIRUS MEDIATED EXPRESSION OF MONOCLONAL ANTIBODY MR191 PROTECTS MICE AGAINST MARBURG VIRUS AND

PROVIDES LONG-TERM EXPRESSION IN SHEEP Article 01 September 2022 REFERENCES * Berger EA, Murphy PM, Farber JM . Chemokine receptors as HIV-1 coreceptors: roles in viral entry, tropism,

disease. _Annu Rev Immunol_ 1999; 17: 657–700. Article  CAS  Google Scholar  * Collman RG, Yi Y . Cofactors for human immunodeficiency virus entry into primary macrophages. _J Infect Dis_

1999; 179: S422–S426. Article  CAS  Google Scholar  * Liu R et al. Homozygous defect in HIV-1 coreceptor accounts for resistance of some multiply-exposed individuals to HIV-1 infection.

_Cell_ 1996; 86: 367–377. Article  CAS  Google Scholar  * Samson M et al. Resistance to HIV-1 infection in caucasian individuals bearing mutant alleles of the CCR-5 chemokine receptor gene.

_Nature_ 1996; 382: 722–725. Article  CAS  Google Scholar  * Dean M et al. Genetic restriction of HIV-1 infection, progression to AIDS by a deletion allele of the CKR5 structural gene.

Hemophilia Growth, Development Study, Multicenter AIDS Cohort Study, Multicenter Hemophilia Cohort Study, San Francisco City Cohort, ALIVE Study. _Science_ 1996; 273: 1856–1862. Article  CAS

  Google Scholar  * Huang Y et al. The role of a mutant CCR5 allele in HIV-1 transmission, disease progression. _Nat Med_ 1996; 2: 1240–1243. Article  CAS  Google Scholar  * Eugen-Olsen J et

al. Heterozygosity for a deletion in the CKR-5 gene leads to prolonged AIDS-free survival, slower CD4 T-cell decline in a cohort of HIV-seropositive individuals. _Aids_ 1997; 11: 305–310.

Article  CAS  Google Scholar  * O'Brien TR et al. 2nd HIV-1 infection in a man homozygous for CCR5 delta 32. _Lancet_ 1997; 349: 1219. Article  CAS  Google Scholar  * Theodorou I et al.

HIV-1 infection in an individual homozygous for CCR5 delta 32. Seroco Study Group. _Lancet_ 1997; 349: 1219–1220. Article  CAS  Google Scholar  * Quillent C et al. HIV-1-resistance

phenotype conferred by combination of two separate inherited mutations of CCR5 gene. _Lancet_ 1998; 351: 14–18. Article  CAS  Google Scholar  * Kupfer B et al. Protection against parenteral

HIV-1 infection by homozygous deletion in the C-C chemokine receptor 5 gene. _Aids_ 1999; 13: 1025–1028. Article  CAS  Google Scholar  * Ometto L et al. Restriction of HIV type 1 infection

in macrophages heterozygous for a deletion in the CC-chemokine receptor 5 gene. _AIDS Res Hum Retroviruses_ 1999; 15: 1441–1452. Article  CAS  Google Scholar  * Ward SG, Bacon K, Westwick J

. Chemokines, T lymphocytes: more than an attraction. _Immunity_ 1998; 9: 1–11. Article  CAS  Google Scholar  * Ward SG, Westwick J . Chemokines: understanding their role in T-lymphocyte

biology. _Biochem J_ 1998; 333: 457–470. Article  CAS  Google Scholar  * Cagnon L, Rossi JJ . Downregulation of the CCR5 beta-chemokine receptor, inhibition of HIV-1 infection by stable

VA1-ribozyme chimeric transcripts. _Antisense Nucleic Acid Drug Dev_ 2000; 10: 251–261. Article  CAS  Google Scholar  * Feng Y et al. Inhibition of CCR5-dependent HIV-1 infection by hairpin

ribozyme gene therapy against CC-chemokine receptor 5. _Virology_ 2000; 276: 271–278. Article  CAS  Google Scholar  * Rossi JJ, Sarver N . RNA enzymes (ribozymes) as antiviral therapeutic

agents. _Trends Biotechnol_ 1990; 8: 179–183. Article  CAS  Google Scholar  * Rossi JJ et al. Ribozymes as therapies for AIDS. _Ann NY Acad Sci_ 1990; 616: 184–200. Article  CAS  Google

Scholar  * Sarver N et al. Ribozymes as potential anti-HIV-1 therapeutic agents. _Science_ 1990; 247: 1222–1225. Article  CAS  Google Scholar  * Steinberger P et al. Functional deletion of

the CCR5 receptor by intracellular immunization produces cells that are refractory to CCR5-dependent HIV-1 infection and cell fusion. _Proc Natl Acad Sci USA_ 2000; 97: 805–810. Article  CAS

  Google Scholar  * Königs C et al. Monoclonal antibody screening of a phage-displayed random peptide library reveals mimotopes of chemokine receptor CCR5: implications for the tertiary

structure of the receptor and for an N-terminal binding site for HIV-1 gp120. _Eur J Immunol_ 2000; 30: 1162–1171. Article  Google Scholar  * Chen JD et al. Inactivation of HIV-1 chemokine

co-receptor CXCR-4 by a novel intrakine strategy. _Nat Med_ 1997; 3: 1110–1116. Article  CAS  Google Scholar  * Strayer DS, Milano J . SV40 mediates stable gene transfer _in vivo_. _Gene

Therapy_ 1996; 3: 581–587. CAS  Google Scholar  * Strayer DS . SV40 as an effective gene transfer vector _in vivo_. _J Biol Chem_ 1996; 271: 24741–24746. CAS  Google Scholar  * Strayer DS,

Kondo R, Milano J, Duan LX . Use of SV40-based vectors to transduce foreign genes to normal human peripheral blood mononuclear cells. _Gene Therapy_ 1997; 4: 219–225. Article  CAS  Google

Scholar  * Strayer DS, Zern MA . Gene delivery to the liver using simian virus 40-derived vectors. _Semin Liver Dis_ 1999; 19: 71–81. Article  CAS  Google Scholar  * Strayer DS . Effective

gene transfer using viral vectors based on SV40. _Methods Mol Biol_ 2000; 133: 61–74. CAS  Google Scholar  * Strayer DS et al. Durability of transgene expression and vector integration:

recombinant SV40-derived gene therapy vectors. _Mol Ther_ 2002; 2: 227–237. Article  Google Scholar  * Kondo R, Feitelson MA, Strayer DS . Use of SV40 to immunize against hepatitis B surface

antigen: implications for the use of SV40 for gene transduction and its use as an immunizing agent. _Gene Therapy_ 1998; 5: 575–582. Article  CAS  Google Scholar  * Strayer DS et al.

Generation of recombinant SV40 vectors for gene transfer. _Methods Mol Biol_ 2001; 165: 103–117. CAS  Google Scholar  * Lee B et al. Epitope mapping of CCR5 reveals multiple conformational

sites and distinct but overlapping structures involved in chemokine and coreceptor function. _J Biol Chem_ 1999; 274: 9617–9626. Article  CAS  Google Scholar  * Goldstein H et al. Gene

therapy using a simian virus 40-derived vector inhibits the development of _in vivo_ human immunodeficiency virus type 1 infection of severe combined immunodeficiency mice implanted with

human fetal thymic and liver tissue. _J Infect Dis_ 2002; 185: 1425–1430. Article  CAS  Google Scholar  * BouHamdan M et al. Inhibition of HIV-1 infection by down-regulation of the CXCR4

co-receptor using an intracellular single chain variable fragment against CXCR4. _Gene Therapy_ 2001; 8: 408–418. Article  CAS  Google Scholar  * Sauter BV et al. A replication-deficient

rSV40 mediates liver-directed gene transfer and a long-term amelioration of jaundice in gunn rats. _Gastroenterology_ 2000; 119: 1348–1357. Article  CAS  Google Scholar  * Signoret N et al.

Differential regulation of CXCR4 and CCR5 endocytosis. _J Cell Sci_ 1998; 111: 2819–2830. CAS  PubMed  Google Scholar  * Means RE et al. Ability of the V3 loop of simian immunodeficiency

virus to serve as a target for antibody-mediated neutralization: correlation of neutralization sensitivity, growth in macrophages, and decreased dependence on CD4. _J Virol_ 2001; 75:

3903–3915. Article  CAS  Google Scholar  * Vincent F et al. Human blood-derived macrophages: differentiation _in vitro_ of a large quantity of cells in serum-free medium. _Exp Hematol_ 1992;

20: 17–23. CAS  PubMed  Google Scholar  * Eischen A et al. Long term cultures of human monocytes _in vitro_. Impact of GM-CSF on survival and differentiation. _J Immunol Methods_ 1991; 143:

209–221. Article  CAS  Google Scholar  * Lee SC, Liu W, Brosnan CF, Dickson DW . Characterization of primary human fetal dissociated central nervous system cultures with an emphasis on

microglia. _Lab Invest_ 1992; 67: 465–476. CAS  PubMed  Google Scholar  * Lee S et al. Coreceptor competition for association with CD4 may change the susceptibility of human cells to

infection with T-tropic and macrophage tropic isolates of human immunodeficiency virus type 1. _J Virol_ 2000; 74: 5016–5023. Article  CAS  Google Scholar  * Wu L et al. CCR5 levels and

expression pattern correlate with infectability by macrophage-tropic HIV-1, _in vitro_. _J Exp Med_ 1997; 185: 1681–1691. Article  CAS  Google Scholar  * Engel BC et al. Intrakines –

evidence for a _trans-_cellular mechanism of action. _Mol Ther_ 2000; 1: 165–170. Article  CAS  Google Scholar  * Strayer DS et al. Combination genetic therapy to inhibit HIV-1. _Mol Ther_

2002; 5: 33–41. Article  CAS  Google Scholar  * Ferkol T, Perales JC, Mularo F, Hanson RW . Receptor-mediated gene transfer into macrophages. _Proc Natl Acad Sci USA_ 1996; 93: 101–105.

Article  CAS  Google Scholar  * Erbacher P et al. Gene transfer by DNA/glycosylated polylysine complexes into human blood monocyte-derived macrophages. _Hum Gene Ther_ 1996; 7: 721–729.

Article  CAS  Google Scholar  * Byrnes AP, Wood MJ, Charlton HM . Role of T cells in inflammation caused by adenovirus vectors in the brain. _Gene Therapy_ 1996; 3: 644–651. CAS  Google

Scholar  * Cremer I, Vieillard V, De Maeyer E . Retrovirally mediated IFN-beta transduction of macrophages induces resistance to HIV, correlated with up-regulation of RANTES production and

down-regulation of C-C chemokine receptor-5 expression. _J Immunol_ 2000; 164: 1582–1587. Article  CAS  Google Scholar  * Naldini L et al. _In vivo_ gene delivery and stable transduction of

nondividing cells by a lentiviral vector. _Science_ 1996; 272: 263–267. Article  CAS  Google Scholar  * Gervaix A et al. Gene therapy targeting peripheral blood CD34+ hematopoietic stem

cells of HIV-infected individuals. _Hum Gene Ther_ 1997; 8: 2229–2238. Article  CAS  Google Scholar  * Davis BR et al. Targeted transduction of CD34+ cells by transdominant negative

Rev-expressing retrovirus yields partial anti-HIV protection of progeny macrophages. _Hum Gene Ther_ 1998; 9: 1197–1207. Article  CAS  Google Scholar  * Junker U et al. Inhibition of human

immunodeficiency virus type 1 replication in myelomonocytic cells derived from retroviral vector-transduced peripheral blood progenitor cells. _Hum Gene Ther_ 1998; 9: 333–340. Article  CAS

  Google Scholar  * Yu M et al. Intracellular immunization of human fetal cord blood stem/progenitor cells with a ribozyme against human immunodeficiency virus type 1. _Proc Natl Acad Sci

USA_ 1995; 92: 699–703. Article  CAS  Google Scholar  * Qin XF, An DS, Chen IS, Baltimore D . Inhibiting HIV-1 infection in human T cells by lentiviral-mediated delivery of small interfering

RNA against CCR5. _Proc Natl Acad Sci USA_ 2003; 100: 183–188. Article  CAS  Google Scholar  * Cordelier P, Morse B, Strayer DS . Targeting CCR5 with siRNAs: using recombinant SV40-derived

vectors to protect macrophages and microglia from R5-tropic HIV. _Oligonucleotides_ 2003; 13: 281–294. Article  CAS  Google Scholar  * Chatterjee S et al. Transduction of primitive human

marrow and cord blood-derived hematopoietic progenitor cells with adeno-associated virus vectors. _Blood_ 1999; 93: 1882–1894. CAS  Google Scholar  * Norkin LC, Anderson HA, Wolfrom SA,

Oppenheim A . Caveolar endocytosis of simian virus 40 is followed by brefeldin A-sensitive transport to the endoplasmic reticulum, where the virus disassembles. _J Virol_ 2002; 76:

5156–5166. Article  CAS  Google Scholar  * Pelkmans L, Kartenbeck J, Helenius A . Caveolar endocytosis of simian virus 40 reveals a new two-step vesicular-transport pathway to the ER. _Nat

Cell Biol_ 2001; 3: 473–483. Article  CAS  Google Scholar  * Parton RG, Lindsay M . Exploitation of major histocompatibility complex class I molecules and caveolae by simian virus 40.

_Immunol Rev_ 1999; 168: 23–31. Article  CAS  Google Scholar  * Norkin LC . Simian virus 40 infection via MHC class I molecules and caveolae. _Immunol Rev_ 1999; 168: 13–22. Article  CAS 

Google Scholar  * Anderson HA, Chen Y, Norkin LC . Bound simian virus 40 translocates to caveolin-enriched membrane domains, and its entry is inhibited by drugs that selectively disrupt

caveolae. _Mol Biol Cell_ 1996; 7: 1825–1834. Article  CAS  Google Scholar  * Paxton WA, Kang S, Koup RA . The HIV type 1 coreceptor CCR5 and its role in viral transmission and disease

progression. _AIDS Res Hum Retroviruses_ 1998; 14: S89–S92. PubMed  Google Scholar  * Aarons EJ et al. Adaptation to blockade of human immunodeficiency virus type 1 entry imposed by the

anti-CCR5 monoclonal antibody 2D7. _Virology_ 2001; 287: 382–390. Article  CAS  Google Scholar  Download references ACKNOWLEDGEMENTS We are grateful to Drs Benhur Lee and Robert W Doms for

arranging to provide CTC8 hybridoma from which SV(2C7) was made. We are also grateful to Dr James A Hoxie for giving us SupT1/CCR5 cells. The technical assistance of Maria Lamothe is

gratefully acknowledged. This work was supported by NIH Grants AI48244, MH70287, MH69122, and RR13156. AUTHOR INFORMATION AUTHORS AND AFFILIATIONS * Department of Pathology, Jefferson

Medical College, Philadelphia, PA, USA P Cordelier, C Ko, A A Matskevitch, H J McKee, G Kari & D S Strayer * Department of Medicine, Jefferson Medical College, Philadelphia, PA, USA J W

Kulkowsky, M Bouhamdan & R J Pomerantz * City of Hope Beckman Institute, Duarte, CA, USA J J Rossi Authors * P Cordelier View author publications You can also search for this author

inPubMed Google Scholar * J W Kulkowsky View author publications You can also search for this author inPubMed Google Scholar * C Ko View author publications You can also search for this

author inPubMed Google Scholar * A A Matskevitch View author publications You can also search for this author inPubMed Google Scholar * H J McKee View author publications You can also search

for this author inPubMed Google Scholar * J J Rossi View author publications You can also search for this author inPubMed Google Scholar * M Bouhamdan View author publications You can also

search for this author inPubMed Google Scholar * R J Pomerantz View author publications You can also search for this author inPubMed Google Scholar * G Kari View author publications You can

also search for this author inPubMed Google Scholar * D S Strayer View author publications You can also search for this author inPubMed Google Scholar RIGHTS AND PERMISSIONS Reprints and

permissions ABOUT THIS ARTICLE CITE THIS ARTICLE Cordelier, P., Kulkowsky, J., Ko, C. _et al._ Protecting from R5-tropic HIV: individual and combined effectiveness of a hammerhead ribozyme

and a single-chain Fv antibody that targets CCR5. _Gene Ther_ 11, 1627–1637 (2004). https://doi.org/10.1038/sj.gt.3302329 Download citation * Received: 09 September 2003 * Accepted: 18 May

2004 * Published: 05 August 2004 * Issue Date: 01 November 2004 * DOI: https://doi.org/10.1038/sj.gt.3302329 SHARE THIS ARTICLE Anyone you share the following link with will be able to read

this content: Get shareable link Sorry, a shareable link is not currently available for this article. Copy to clipboard Provided by the Springer Nature SharedIt content-sharing initiative

KEYWORDS * rSV40 * CCR5 * intrabody * intrakine * macrophages * microglia