Inhibition of human immunodeficiency virus type 1 replication in vitro in acutely and persistently infected human CD4+ mononuclear cells expressing murine and humanized anti-human immunodeficiency virus type 1 Tat single-chain variable fragment intrabodies.
We have previously reported that a murine anti-Tat sFv intrabody, termed sFvtat1Ck, directed against the proline-rich N-terminal activation domain of HIV-1, is a potent inhibitor of HIV-1 replication [Mhashilkar, A. M., et al. (1995). EMBO J. 14, 1542-1551]. In this study, the protective effect of sFvtat1Ck expression on HIV-1 replication in both acutely infected and persistently infected CD4+ cells was examined. Stably transfected CD4+ SupT1 cells were resistant to HIV-1 infection at high MOI with both the laboratory isolate HxB2 and six syncytium-inducing (SI) primary isolates. Persistently infected U1 cells, which can be induced to increase HIV-1 mRNA synthesis on addition of PMA or TNF-alpha, showed decreased production of HIV-1 in the presence of sFvtat1Ck. In transduced CD4+-selected, CD8+-depleted, and total PMBCs, the sFvtat1Ck-expressing cells showed marked inhibition of HIV-1 replication. The anti-Tat sFv was subsequently humanized by substituting compatible human framework regions that were chosen from a large database of human V(H) and V(L) sequences on the basis of high overall framework matching, similar CDR length, and minimal mismatching of canonical and V(H)/V(L) contact residues. One humanized anti-Tat sFv intrabody, termed sFvhutat2, demonstrated a level of anti-HIV-1 activity that was comparable to the parental murine sFv when transduced PBMCs expressing the murine or humanized sFv intrabodies were challenged with HxB2 and two SI primary isolates. Because Tat is likely to have both direct and indirect effects in the pathogenesis of AIDS through its multiple roles in the HIV-1 life cycle and through its effects on the immune system, the strategy of genetically blocking Tat protein function with a humanized anti-Tat sFv intrabody may prove useful for the treatment of HIV-1 infection and AIDS, particularly when used as an adjuvant gene therapy together with highly active antiretroviral therapies that are currently available.
Department of Pathology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA 02115, USA.
A M
AM
Mhashilkar
J
J
LaVecchio
B
B
Eberhardt
J
J
Porter-Brooks
S
S
Boisot
J H
JH
Dove
C
C
Pumphrey
X
X
Li
R N
RN
Weissmahr
D B
DB
Ring
U
U
Ramstedt
W A
WA
Marasco
D000595:Amino Acid Sequence; D000818:Animals; D015496:CD4-Positive T-Lymphocytes / Q000276:immunology* / Q000821:virology; D015696:Gene Products, tat / Q000276:immunology*; D018014:Gene Transfer Techniques; D015726:Giant Cells; D015483:HIV Antibodies / Q000235:genetics / Q000276:immunology*; D015497:HIV-1 / Q000276:immunology* / Q000502:physiology; D006801:Humans; D007128:Immunoglobulin Fragments / Q000235:genetics / Q000276:immunology*; D007135:Immunoglobulin Variable Region / Q000235:genetics / Q000276:immunology*; D007963:Leukocytes, Mononuclear / Q000166:cytology / Q000821:virology; D051379:Mice; D008969:Molecular Sequence Data; D009000:Monocytes / Q000166:cytology / Q000821:virology; D014162:Transfection; D014407:Tumor Cells, Cultured; D014779:Virus Replication; D054322:tat Gene Products, Human Immunodeficiency Virus
D015696:Gene Products, tat; D015483:HIV Antibodies; D007128:Immunoglobulin Fragments; D007135:Immunoglobulin Variable Region; D054322:tat Gene Products, Human Immunodeficiency Virus