DNA immunization with HIV-1 tat mutated in the trans activation domain induces humoral and cellular immune responses against wild-type Tat.

Intramuscular immunization of mice with plasmids encoding two transdominant negative mutants of the HIV-1 Tat protein (Tat22 and Tat22/37) elicited a humoral response to wild-type Tat that is comparable to that induced by inoculation of wild-type tat DNA or Tat protein. The percentage of the responders and the Ab titers continued to increase after three additional DNA boosts and pretreatment with bupivacaine at the site of inoculation, without a significant difference (p > 0.05) among the three groups of mice immunized with mutant and wild-type tat genes. By utilizing synthetic peptides representing the amino acid sequence of Tat, one major B cell epitope was defined within the cysteine-rich domain of Tat. Anti-Tat IgG Abs directed against this epitope were found in mice immunized with all tat DNA constructs, whereas different Tat epitopes were detected in mice immunized with the Tat protein. Similarly, IgG2a was the predominant isotype in DNA-immunized mice, with both mutants and wild-type tat genes, as compared with protein immunization, which induced mostly IgG1 and IgG3. Sera from most immunized mice neutralized the effect of extracellular Tat in activating HIV-1 replication. A cellular response was also elicited as indicated by the proliferation of splenocytes when stimulated with wild-type Tat. These results indicate that the wild-type Tat Ag is recognized by Abs and T cells induced by DNA immunization with mutated tat genes, suggesting the possible use of these Tat transdominant mutants, lacking viral trans activation activity and capable of blocking wild-type Tat activity, in the development of an anti-HIV-1 vaccine.

Studies on the effect of the combined expression of anti-tat and anti-rev genes on HIV-1 replication.

A series of retroviral vectors with potential anti-tat and antirev activity was developed. Vectors containing a tat transdominant negative mutant (tat22/37) and an RRE decoy in different positions, directed by the same promoter or by different promoters, were generated. Retroviral vectors containing tat22/37 and the RevM10 transdominant negative mutant were also constructed. Jurkat cells were transduced with the recombinant retroviruses to produce monoclonal and polyclonal cultures. In these cell lines the recombinant proviruses were correctly integrated and expression of the inserted genes was detected by Northern blot or RT-PCR analysis. However, infection of these cell lines with HIV-1 showed that none of these recombinant constructs inhibited virus replication at a high multiplicity of infection (MOI). At a low MOI, two cell clones containing tat22/37 and the RRE decoy in 3' position showed a long lasting protection against virus replication, in comparison to control cultures expressing tat22/37 or RRE alone. Combination of tat and rev mutants was ineffective in inhibiting HIV-1 replication at both low and high MOIs. At a low MOI, HIV-1 replication was efficiently blocked in two cell clones expressing the RevM10 mutant alone. These results show a synergic effect of anti-tat and anti-rev molecules when the RRE sequence is cloned 3' to tat22/37, suggesting the possibility of using this vector design to control HIV-1 replication.

Red blood cells as delivery system for recombinant HSV-1 glycoprotein B: immunogenicity and protection in mice.

The immunotherapeutic potential of autologous red blood cells (RBC) coupled to the secretory form of herpes simplex virus type 1 (HSV-1) glycoprotein B (gB1s) was examined with a mouse model of HSV-1 infection. C57BL/6 mice were immunized intraperitoneally with gB1s (0.05 microgram per dose) linked to RBC, or mixed with Freund's complete or bound to AlPO4 adjuvants (0.5 microgram per dose). Mice immunized with RBC coupled gB1s were protected against lethal and latent HSV-1 infection, and developed an anti-HSV antibody response, as measured by ELISA and HSV-1 neutralization assays, similar or higher than that elicited by the same antigen in Freund's complete adjuvant, which suggested that autologous RBC coupled to gB1s may provide an effective and safe method of immunization against HSV infection.

Inhibition of HIV-1 replication by a Tat transdominant negative mutant in human peripheral blood lymphocytes from healthy donors and HIV-1-infected patients.

It was previously shown that a tat mutant (tat22) where cysteine 22 is substituted by glycine behaves as a transdominant negative mutant in Jurkat T cells lytically or latently infected by HIV-1. In this study we demonstrate that tat22 controls HIV-1 replication in primary cells. This effect was observed both after in vitro infection of peripheral blood mononuclear cells (PBMCs) from normal donors and after reactivation of the latent infection in PBMCs from seropositive patients. The antiviral effect of tat22 was limited to conditions of low virus production. The use of tat22 may be promising for a gene therapy approach to AIDS during the asymptomatic phase of the disease allowing control of virus replication in infected cells and inhibition of virus spread to uninfected cells.

Inhibition of HIV-1 replication and reactivation from latency by tat transdominant negative mutants in the cysteine rich region.

Tat mutants (tat22, tat37 and tat22/37) were constructed in the transactivation domain, where cysteines at positions 22 or/and 37 were substituted with glycine and serine, respectively. These mutants were expressed either in a BK virus episomal vector or in the retroviral vector LXSN. Constitutive production of tat22 by Jurkat T cells in the context of both vectors blocked HIV-1 replication during lytic infection. Conversely, the tat37 mutant did not show any inhibitory activity and tat22/37 displayed a mild effect on HIV-1 infection only when expressed by the recombinant retrovirus. However, constitutive production of tat22/37 by the BK virus vector in Jurkat T cells chronically infected by HIV-1 was effective in blocking reactivation of viral replication induced by tumor necrosis factor-alpha or human herpesvirus-6. These results suggest that mutants in the transactivation domain of tat may be considered in designing alternative strategies to control HIV-1 replication and reactivation from latency during different phases of infection.

Inhibition of human immunodeficiency virus reactivation from latency by a tat transdominant negative mutant.

A BK virus (BKV) expression vector, specific for human cells, was engineered to express antisense human immunodeficiency virus type 1 (HIV-1) tat cDNA (tat-AS) or a tat mutant in cysteine 22 (tat22). Cysteine residues in the cysteine-rich domain of tat are necessary for tat transactivation of the HIV-1 long terminal repeat (LTR). Both the AS tat and the tat mutant significantly inhibited transactivation by tat when assayed in cells cotransfected with an expression vector where the reporter gene for chloramphenicol acetyl transferase was driven by the HIV-1 LTR. Infection of Jurkat cell clones stably expressing tat22 (Jurkat/tat22) or tat-AS (Jurkat/tat-AS) with HIV-1 did not show differences in virus titer in comparison to HIV-1-infected control cells. However, in two Jurkat/tat22 cell clones, entrance of HIV-1 into latency was accelerated significantly and reactivation of HIV-1 from latency induced by tumor necrosis factor-alpha (TNF-alpha) or tat was blocked. These results suggest that, in a combined and integrated approach to the treatment of acquired immunodeficiency syndrome (AIDS), anti-tat genetic therapy could be successfully applied to maintain virus in latency, thereby extending the duration of the asymptomatic phase preceding full-blown AIDS.

High expression of exogenous cDNAs directed by HIV-1 long terminal repeat in human cells constitutively producing HIV-1 tat and adenovirus E1A/E1B.

A eukaryotic vector-host cell system is described where the additive transactivating effects of HIV-1 tat and adenovirus E1A on HIV-1 long terminal repeat (LTR) are exploited to increase expression of exogenous cDNAs. Human 143B and 293 cells, the latter constitutively producing E1A, were used as host cell lines. The bacterial gene chloramphenicol acetyltransferase (CAT) and the hepatitis B surface antigen (HBs-Ag) gene were employed as reporter genes inserted in pRPneoU3R, an episomal vector containing BK virus replication origin and early region, where cDNAs are expressed under control of HIV-1 LTR. The 293 cells were transformed by tat expression vectors to constitutively express tat. Stable cell clones of 293tat cells, constitutively expressing CAT after transformation with pRPneoU3R-CAT, show a CAT activity 600-fold higher than normal 293 transformed cells. CAT expression obtained in normal 293 cells can be transiently increased 10-fold by transfection by vectors expressing tat. The 293tat cells transformed by pRPneoU3R-HBs, an episomal vector expressing HBs-Ag from HIV LTR, yielded stable cell clones secreting HBs-Ag in the culture medium at a concentration up to 744 ng/ml or 44 ng/10(6) cells/24 h, 48-fold more than normal 293 cells. The use of this system for constitutive or inducible expression of sequences under control of HIV-1 LTR is discussed in view of possible applications for diagnostic, vaccinal and therapeutic purposes.

Protection from herpes simplex virus type 1 lethal and latent infections by secreted recombinant glycoprotein B constitutively expressed in human cells with a BK virus episomal vector.

The herpes simplex virus type 1 (HSV-1) glycoprotein B (gB-1) gene, deleted of 639 nucleotides that encode the transmembrane anchor sequence and reconstructed with the extramembrane and intracytoplasmic domains, was cloned under control of the Rous sarcoma virus long terminal repeat in the episomal replicating vector pRP-RSV, which contains the origin of replication and early region of the human papovavirus BK as well as a cDNA for a mutant mouse dihydrofolate reductase that is resistant to methotrexate. gB-1 (0.15 to 0.25 pg per cell per 24 h) was constitutively secreted into the culture medium of pRP-RSV-gBs-transformed human 293 cells. Treatment of transformed cells with methotrexate at high concentrations (0.6 to 6 microM) increased gB-1 production 10- to 100-fold, because of an amplification of the episomal recombinant. Mice immunized with secreted gB-1 produced HSV-1- and HSV-2-neutralizing antibodies and were protected against HSV-1 lethal, latent, and recurrent infections. Constitutive expression of secreted gB-1 in human cells may establish a system to develop diagnostic material and a subunit vaccine for HSV infections.

Suppression of BK virus replication and cytopathic effect by inhibitors of prokaryotic DNA gyrase.

Nalidixic acid and oxolinic acid, two antibacterial agents known to inhibit bacterial DNA gyrase, are shown to suppress the replication, as well as the cytopathic effect, of BK virus in Vero cell cultures. The inhibition of virus replication was detectable at day 4 post infection in cultures which had been continuously exposed to drugs at concentrations as low as 0.02 to 0.04 mM of nalidixic acid and 0.2 mM of oxolinic acid. These active concentrations are inferior to plasma levels attained in the course of clinical use of the drugs for antibacterial chemotherapy. Also, under these circumstances, no cytotoxicity occurred. The inhibition of development of cytopathology and of virus-induced cell death was demonstrable in cultures treated for 12 days with the drugs. Under these circumstances of prolonged action, oxolinic acid proved to be slightly cytotoxic in that virus inhibitory doses reduced the viability of normal cells. No alterations in the topological conformation of the viral genome or accumulation of end products of viral DNA replication were detected. However, accumulation of viral DNA form I at 48 h post infection suggests that the drugs act through a mechanism involving DNA topoisomerase.

Factors affecting amplification of BK virus episomal vectors in human cells. Brief report.

Analysis of factors determining replication of BK virus (BKV) episomal vectors in human cells showed that vector copy number was related to the level of BKV T antigen expression. T antigen was synthesized efficiently, as assessed by indirect immunofluorescence, in vector-transfected primary embryonic fibroblasts undergoing neoplastic transformation. Surprisingly, transfected continuous cell lines (143 B, HeLa and KB), kept under biochemical selection or tested in transient assays, produced negligible amounts or no T antigen, revealed only by a sensitive ELISA test, suggesting that in these cells vector amplification was under the control of cellular factors. Presence or absence of BKV late region sequences, BKV strain, orientation of the inserted genes and presence or absence of selection were not relevant for vector replication. Type of biochemical selection, however, was important, since BKV vectors containing the thymidine kinase gene replicated better than those containing the neo gene. Despite great variability, vector copy number increased in transfected clones of adenovirus 5-transformed 293 cells, in the absence of immunofluorescence detectable T antigen. These cells express adenovirus immediate early proteins E1A and E1B which may directly or indirectly activate BKV origin of replication.

New BK virus episomal vector for complementary DNA expression in human cells.

The properties of pRP-c, a new vector for complementary DNA (cDNA) expression, are described. The vector contains the early region and replication origin of BK virus (BKV), a human papovavirus. Due to the presence of these BKV sequences, pRP-c replicates in human cells allowing amplification of inserted cDNAs. The promoter, intron and polyadenylation region for cDNA expression are separated by unique restriction sites and can therefore be individually excised and substituted with different transcription signals. Coding sequences of the bacterial genes for chloramphenicol-acetyl transferase (CAT) or neomycin phosphotransferase (neo) were inserted into the cDNA cloning site of pRP-c and expressed in human cells in transient assays or stable clones. In both cases expression of the inserted sequences was significantly more efficient than by using the integration vectors pSV2CAT and pSV2neo, demonstrating the advantages of episomal expression vectors in human cells. Possible uses of pRP-c to express viral and cellular cDNAs in human cells are discussed.

Restricted replication of BK virus in human lymphocytes.

Human lymphocytes from either peripheral blood or continuous cultures (P3HR-1 cells) are able to support the replication of prototype polyomavirus BK (BKV) as well as its related strain BO15 virus (BO15V). Instead, human monocytes from peripheral blood, although able to bind and egulf BKV virions, do not express virus-specific antigens within a 50 day observation period. In the light of these results, a probable role is suggested for human mononuclear blood cells in the mechanism of natural infection by polyomaviruses.

BK virus-plasmid expression vector that persists episomally in human cells and shuttles into Escherichia coli.

We describe a novel expression vector, pBK TK-1, that persists episomally in human cells that can be shuttled into bacteria. This vector includes sequences from BK virus (BKV), the thymidine kinase (TK) gene of herpes simplex virus type 1, and plasmid pML-1. TK+-transformed HeLa and 143 B cells contained predominantly full-length episomes. There were typically 20 to 40 (HeLa) and 75 to 120 143 B vector copies per cell, although some 143 B transformants contained hundreds. Low-molecular-weight DNA from TK+-transformed cells introduced into Escherichia coli were recovered as plasmids that were indistinguishable from the input vector. Removal of selective pressure had no apparent effect upon the episomal status of pBK TK-1 molecules in TK+-transformed cells. BKV T antigen may play a role in episomal replication of pBK TK-1 since this viral protein was expressed in TK+ transformants and since a plasmid that contained only the BKV origin of replication was highly amplified in BKV-transformed human cells that synthesize BKV T antigen.

Episomal DNA of a BK virus variant in a human insulinoma.

BK virus (BKV) DNA was detected by blot hybridization in a human adenoma of pancreatic islets from patient I.R. BKV DNA was free, and no evidence was found of viral sequences integrated into cellular DNA. Virus was rescued by transfection of human embryonic fibroblasts with tumor DNA. The DNA from rescued virus (BKV-IR) was different from wild-type BKV DNA by restriction endonuclease mapping. The genome of BKV-IR is 235 base pairs (bp) shorter than the genome of wild-type BKV. This alteration originates from a deletion of approximately 300 bp involving HindIII fragments B and D, and an insertion of 70 bp in the region of HindIII fragment C. Transformation of hamster kidney cells was induced by total tumor DNA as well as by BKV-IR and BKV-IR DNA. No antibodies to BKV tumor (T) antigen were detected in the patient's serum by immunofluorescence. The significance of episomal BKV DNA in a human tumor is discussed.

Formation of oligomeric free BK virus DNA is required for tandem integration of viral genomes into cellular DNA.

Mouse kidney cells have been transformed by linear BK virus (BKV) DNA with cohesive ends. These BKV genomes can circularize and subsequently replicate or physically join end to end yielding oligomeric viral DNA molecules. Blot hybridization analysis of transformed cells showed the presence of BKV DNA both in free circular forms and integrated into cellular DNA in a tandem head-to-tail array of full-length viral genomes. Formation of oligomers either by replication via previous circularization or by end to end joining is hindered after elimination of cohesive termini by digestion with the single-strand-specific nuclease S1. After treatment of linear BKV DNA with nuclease S1 only integrations of single viral genomes were observed in transformed cells; tandem insertions and free viral DNA were absent. These results support the hypothesis that formation of tandems of free viral DNA is a necessary prerequisite for tandem integration of viral genomes.

Transformation of human embryonic fibroblasts by BK virus, BK virus DNA and a subgenomic BK virus DNA fragment.

Human embryonic fibroblasts (HEF) have been transformed by BK virus (BKV) DNA and by u.v.-inactivated or live BKV alone or in association with methyl-cholanthrene (MTC). The transformed cells produced BKV large T and small t antigens as well as the cellular 53 kdal protein, detected by immunofluorescence and immunoprecipitation. After an initial phase of lysis and virus shedding, virus or its coat protein antigen could not be detected in transformed cells. All human transformed cell lines could be superinfected by BKV or BKV DNA, but their susceptibility to superinfection was 20- to 500-fold lower than normal HEF. BKV could be rescued by fusion of transformed cells with normal HEF or Vero cells and by transfection of normal HEF with total DNA and DNA extracted from the Hirt supernatant of transformed cells. Blot hybridization analysis of DNA from transformed cells showed a considerable amount of free BKV DNA in monomeric and polymeric forms. Integrated BKV DNA was absent in most cell lines but present in only small amounts in BKV-transformed cells treated with MTC. Analysis of free BKV DNA with various restriction endonucleases and by blot hybridization showed that monomeric forms were complete BKV genomes, whereas polymers contained both complete and defective or rearranged BKV DNA. Transformation of HEF was also obtained with a 3.7 kilobase (kb) fragment of the BKV genome, produced by sequential digestion of BKV with the restriction endonucleases HhaI and EcoRI. This fragment extends clockwise on the virus genome from 0 to 72.2 map units and contains the entire early region. Blot hybridization analysis of cells transformed by the HhaI/EcoRI 3.7 kb fragment showed two separate integrations of BKV sequences without free virus DNA.