Increased encapsulated cell biodelivery of nerve growth factor in the brain by transposon-mediated gene transfer.

Nerve growth factor (NGF) is a potential therapeutic agent for Alzheimer's disease (AD) as it has positive effects on the basal forebrain cholinergic neurons whose degeneration correlates with the cognitive decline in AD. We have previously described an encapsulated cell biodelivery device, NsG0202, capable of local delivery of NGF by a genetically modified human cell line, NGC-0295. The NsG0202 devices have shown promising safety and therapeutic results in a small phase 1b clinical study. However, results also show that the NGF dose could advantageously be increased. We have used the sleeping beauty transposon expression technology to establish a new clinical grade cell line, NGC0211, with at least 10 times higher NGF production than that of NGC-0295. To test whether encapsulation of this cell line provides a relevant dose escalation step in delivering NGF for treatment of the cognitive decline in AD patients, we have validated the bioactivity of devices with NGC0211 and NGC-0295 cells in normal rat striatum as well as in the quinolinic acid striatal lesion model. These preclinical animal studies show that implantation of devices with NGC0211 cells lead to significantly higher NGF output, which in both cases correlate with highly improved potency.

Role of the TATA box in transcription of the mouse mammary tumor virus long terminal repeat.

An in vitro transcription system from mammary cells was established to study transcription of the long terminal repeat (LTR) of the mouse mammary tumor virus (MMTV). Experiments with progressive 5'-deletion constructs of the MMTV LTR revealed that a 19-base pair (bp) region from -41 to -23 bp, encompassing the TATA box and flanking DNA sequence, was as transcriptionally active as larger promoter constructs, both in nuclear extracts from human mammary cell lines (T47D and MCF7) and a nonmammary cell line (HeLa). The cell-free system was capable of supporting transcriptional induction by factors binding upstream of the TATA box, however, since purified glucocorticoid receptor-induced transcription in larger promoter constructs encompassing the MMTV hormone-responsive elements. Transcription from two other promoters, the adenovirus major late promoter and the human immunodeficiency virus LTR, also revealed a significant transcriptional contribution of upstream elements. The 19-bp TATA region from the MMTV LTR was shown to have considerably more activity in this transcription system than comparable TATA regions from other promoters. Sequences critical to the MMTV TATA region were evaluated by single base pair mutagenesis and found to comprise a consensus TATA box sequence, TATAAAA, as well as a single A just upstream of the TATAAAA sequence. Thus, the high level of basal transcription observed with the TATA region from MMTV is due to a perfect consensus TATA box sequence and a single base immediately 5' adjacent. It is likely that the high basal rate of transcription observed with this TATA box region on histone-free templates represents an inappropriate level of basal expression and that a complete evaluation of transactivation mechanisms in this system will require the recapitulation in vitro of the chromatin-mediated repressive state that exists in vivo.

Mapping of two new human B-cell epitopes on HIV-1 gp120.

OBJECTIVE: To map epitopes on gp120 defined by human antibodies and to examine the neutralizing activity of these antibodies. DESIGN AND METHODS: Serum from HIV-1-antibody-positive individuals was used to screen a random fragment expression library representing gp120 from the HIVIIIB clone BH10. The library was based on the pUEX1 expression vector. Serum was tested for in vitro neutralizing activity using H9 cells and the HIVIIIB isolate. RESULTS: Four different epitopes defined by human antibodies were mapped on gp120. Two of these have not previously been reported and are located within amino acids (aa) 90-100 in the C1 region and aa 355-365 in the semi-conserved region between V3 and V4. The other two are located within aa 140-145 and aa 286-309. These epitopes are situated in regions that have been shown to demarcate human epitopes. Three serum samples with neutralization titres > or = 1024 were identified. None of the purified antibody fractions defining the mapped epitopes on gp120 had any neutralizing capacity against HIVIIIB. CONCLUSIONS: This study is the first demonstration of the applicability of random fragment expression libraries for the direct screening of human serum in order to map epitopes on gp120. Two new epitopes and two previously identified epitopes were mapped in this way. However, none of the linear epitopes was defined by antibody fractions neutralizing HIVIIIB, and it was not possible to map epitopes defined by neutralizing antibodies in the serum samples capable of neutralizing HIVIIIB infection of H9 cells. Thus, it appears that the neutralizing activity of serum in this study was not due to anti-gp120 antibodies defining linear epitopes.

Immunological characterization and detection of the major core protein p24 of the human immunodeficiency virus (HIV) using monoclonal antibodies.

Eight different monoclonal antibodies (MAbs) were raised against a lysate of the HTLV-IIIb isolate of human immunodeficiency virus (HIV). All eight MAbs recognized the major core protein p24 as well as the gag precursors p39 and p55. Three different epitopes were defined by the eight MAbs when an antigen-catching ELISA was used as the test system. An antigen-catching ELISA for p24 was developed by use of two of the MAbs defining two different epitopes. This ELISA system was applied to the detection of p24 in culture supernatants from lymphocyte cultures of 13 different HIV isolates. The present p24 detecting ELISA proved useful for characterization of different isolates of HIV. Further, two MAbs from the present panel of antibodies were demonstrated to be sensitive and specific probes for the immunohistological detection of p24 protein in tissue sections of lymphoid tissue.

Mapping of a new immunodominant human linear B-cell epitope on the vpu protein of the human immunodeficiency virus type 1.

A random fragment expression library was used to identify and map a new human epitope on the vpu protein of the human immunodeficiency virus type 1 (HIV-1). The epitope was mapped to the central part of the protein within amino acids (aa) 37-50 comprising the sequence N-KIDRLIDRLI-ERAE-C. A alpha-galactosidase-vpu fusion protein representing aa 37-68 of vpu was used to screen 356 human serum samples from HIV-1-infected persons for antibodies to the novel epitope. A total of 125 (35.1%) of the samples reacted with this region of vpu. Antibodies against this region were significantly more prevalent among samples from individuals with CD4 cell counts < 400 cells/microliters than individuals with CD4 cell counts > or = 400 cells/microliters (37.6 vs. 17.6%; p < 0.0146, Fisher's exact test). Thus, the presence of antibodies against this epitope of vpu appears to be associated with a progressed state of disease.

Analysis of the secondary structure of the human immunodeficiency virus (HIV) proteins p17, gp120, and gp41 by computer modeling based on neural network methods.

A neural network computer program, trained to predict secondary structure of proteins by exposing it to matching sets of primary and secondary structures from a database, was used to analyze the human immunodeficiency virus (HIV) proteins p17, gp120, and gp41 from their amino acid sequences. The results are compared to those obtained by the Chou-Fasman analysis. Two alpha-helical sequences corresponding to the putative fusigenic domain and to the transmembrane domain of gp41 could be predicted, as well as a possible binding site between p17 and gp41. On the basis of the secondary structure predictions, a three-dimensional model of p17 was constructed. This model was found to represent a stable conformation by an analysis using an energy-minimization program. The model predicts that p17 is attached to the membrane only by the acylated N-terminus, in analogy with the N-terminus of the gag protein of other retroviruses and also with the src oncogene protein p60src. The intracellular C-terminal part of gp41 may act as a receptor by electrostatic interaction with p17.

Mapping of linear B-cell epitopes on the major core protein p24 of human immunodeficiency virus type 1 (HIV-1).

Three murine monoclonal antibodies (MAbs) F5-2, F5-4, and F5-16 defining three different epitopes on the major core protein p24 of the human immunodeficiency virus type 1 (HIV-1) were epitope mapped using a random fragment expression library representing the p17- and p24-encoding part of the gag open reading frame. F5-2 defined an epitope within amino acids (aa) 14-23 at the N-terminus of p24, and F5-4 defined an epitope within aa 153-174 in the C-terminus of p24. F5-16 did not recognize any of the fusion proteins produced by the expression library indicating that this MAb defines a true conformational epitope on p24. Since the N-terminus of p24 has been reported to be immunosilent in humans, 356 HIV-1 antibody-positive serum samples were tested for reactivity against the region of p24 defined by F5-2. More than one third of the samples recognized this region indicating that it is immunoreactive and, further, the presence of antibodies against this region was associated with a reduced CD4 cell count.

A comparison of three methods for detection of antibodies against the major core protein p24 of human immunodeficiency virus.

The native major core protein p24 of the human immunodeficiency virus (HIV) was immunoaffinity purified by a monoclonal antibody and used to develop an indirect enzyme-linked immunosorbent assay (inELISA) for detecting p24 antibodies in human sera. Its ability to detect p24 antibodies was compared to that of the immunoblotting test (IBT) and a commercial available competition ELISA (compELISA) employing recombinant HIV core protein. In tests on 60 serum samples the overall agreement of the inELISA and the IBT was 93.3%. Fifty-two samples were p24 antibody positive in both the inELISA and the IBT and of these 24 (46.2%) were positive in the compELISA. All compELISA positive samples were derived from healthy individuals, whereas of the 28 (53.8%) compELISA negative samples 1 was from a patient with acute HIV infection, 18 from healthy individuals and 9 from ARC/AIDS patients. The compELISA was able to distinguish among healthy persons with normal or low T-helper cell count (P = 0.048), as was the inELISA when p24 antibodies were titrated (P = 0.027). The inELISA equals IBT in specificity and sensitivity, is convenient and is very suitable for titration of p24 antibodies.

Competition ELISA using a human monoclonal antibody for detection of antibodies against human immunodeficiency virus type 1.

A novel competition ELISA for detection of antibodies against HIV-1 was developed. The assay is based on competition at the single epitope level and utilises a human monoclonal antibody and an E. coli-produced fragment of the transmembrane glycoprotein gp41. The sensitivity of the assay was 100% in tests on 247 serum samples obtained from 219 individuals previously shown to be HIV-1 antibody positive by both conventional indirect ELISA and the immunoblotting test. The patients represented various clinical and immunological stages of HIV-1 infection. Likewise, the specificity of the assay was 100% in tests on 105 serum samples from normal individuals previously tested negative by indirect ELISA. Further, among 105 serum samples selected due to consistent false positive reactions in the indirect ELISA only 2 samples (1.9%) demonstrated false positive reactions in the competition ELISA, i.e. 98.1% specificity. Finally, only 2 of 57 (3.5%) serum samples from HIV-2 infected individuals showed positive reactions in the assay, while 54 (94.7%) had absorbance values similar to the negative controls. These results demonstrate that human monoclonal antibodies may form the basis for highly sensitive and specific assays for detection of antibodies to HIV-1.

Serological response in patients with chronic asymptomatic human immunodeficiency virus infection.

To examine a possible relationship between decreased immune function and serological parameters, such as human immunodeficiency virus (HIV) antigenaemia and the quality and quantity of whole virus antibodies and antibodies against the major core protein p24, we investigated 160 healthy HIV infected individuals (CDC classification II and III). According to the number of T-helper lymphocytes (CD4 cells) these were divided into two groups (CD4 cell counts above or below 500/microliter), which according to the lymphocyte transformation response to pokeweed mitogen (response above or below 20% of control value) were further subdivided into two groups. Both the presence of HIV antigen (p = 0.022) and the absence of p24 antibodies (p = 0.001) correlated to a decreased CD4 cell count. Lack of p24 antibodies was more frequent than was the presence of HIV antigen among persons with decreased CD4 cell count and decreased response to pokeweed mitogen, indicating that absence of p24 antibodies may be an earlier marker of immune dysfunction than the presence of HIV antigen. In persons with p24 antibodies present, a low such titer was associated with a decrease of both immune parameters. Presence of HIV antigen and absence of p24 antibodies thus seems to correlate with the severity of immune dysfunction in healthy HIV infected individuals.

Characterization of an NF-1/CTF family member as a functional activator of the mouse mammary tumor virus long terminal repeat 5' enhancer.

The long terminal repeat of the mouse mammary tumor virus restricts virus expression primarily to the mammary epithelium. The extreme 5' end of the long terminal repeat contains an enhancer that has been associated with tissue-specific expression of the virus. A total of six functional cis-acting elements have been identified in the enhancer. Although proteins binding to these elements have been reported, only one has been identified; this factor, mp5, is identical or closely related to the transcription factor AP-2 (Mellentin-Michelotti, J., John, S., Pennie, W. D., Williams, T., and Hager, G. L. (1994) J. Biol. Chem. 269, 31983-31990). The other factors are hitherto unidentified and poorly described. We report here the characterization of another of the six elements, previously referred to as the F3 site (Mink, S., Hartig, E., Jennewein, P., Doppler, W., and Cato, A. C. (1992) Mol. Cell Biol. 12, 4906-4918). We show that the F3 binding activity and AP-2 act synergistically to enhance mouse mammary tumor virus-directed transcription, but only in the presence of glucocorticoid hormone. The F3 element has an NF-1-like half-site, but the activity recognizing this element has binding characteristics distinct from the NF-1/CTF family as well as the rest of the CCAAT-binding proteins. We conclude that the F3 activity represents a new member of the NF-1/CTF family.

Purification and stabilization of transcriptionally active glucocorticoid receptor.

A major obstacle to the purification of glucocorticoid receptor (GR) is the very high nonspecific surface adsorption of this protein. This phenomenon is a property of the GR itself and does not reflect overall protein concentration or buffer conditions. We have observed that the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid (CHAPS) is unique in its ability to stabilize the receptor and largely eliminate loss to nonspecific adsorption. We have coupled this observation with a two-step purification method that allows efficient purification and stabilization of transcriptionally active glucocorticoid receptor. For this procedure, the GR first undergoes a major purification by anion exchange chromatography following hormone binding and on-column receptor transformation. Second, the GR is resolved to homogeneity utilizing a hydrophobic interaction chromatography step which consists of a 2.5 M to 0 M NaCl gradient elution of contaminating proteins followed by displacement of GR by CHAPS. GR at both stages of purification was able to activate transcription from the glucocorticoid response element containing the promoter region of the long terminal repeat of the mouse mammary tumor virus. This simple and efficient methodology should be of a considerable advantage for studies of the biology of the active, full-length GR.

Autologous antibody response against the principal neutralizing domain of human immunodeficiency virus type 1 isolated from infected humans.

High titers of neutralizing antibodies in human immunodeficiency virus type 1 (HIV-1) infection are directed primarily against the third hypervariable domain (V3) of the virion envelope glycoprotein gp120. This region has been designated the principal neutralizing domain of HIV-1. Because the frequency and significance of autologous V3 antibodies in natural infection are not fully clarified, we have cloned, sequenced, and expressed the V3 domain from virus of HIV-1-infected patients to test the autologous and heterologous V3 antibody response. The resulting recombinant Escherichia coli V3 fusion proteins reacted strongly with both autologous and heterologous patient antibodies in Western blots. Thirty-one different V3 fragments were cloned from 24 hemophiliac patients with different immunological and clinical statuses. Antibody reactivity against the autologous V3 fusion proteins was detected in all serum samples except one; moreover, all serum samples contained antibody reactivity against a vast majority of heterologous fusion proteins despite significant amino acid variability in V3. The results suggest that V3 antibodies are highly prevalent; further, we find no association between the stage of the HIV-1 infection and the presence of V3 antibodies.

Analysis of a highly immunodominant epitope in the human immunodeficiency virus type 1 transmembrane glycoprotein, gp41, defined by a human monoclonal antibody.

A human monoclonal antibody, 41-7 [immunoglobulin G1(kappa)], directed against the transmembrane glycoprotein gp41 of the human immunodeficiency virus type 1 (HIV-1) has been produced by direct fusion of lymph node cells from an HIV-1-infected individual with a human B-lymphoblastoid cell line. The minimal essential epitope for 41-7 was mapped to a conserved seven-amino acid sequence, N-CSGKLIC-C, located within the N-terminal part of gp41. Antibodies blocking the binding of 41-7 could be detected in the serum of all HIV-1-infected individuals tested, irrespective of the stage of the infection. The epitope is located externally to the plasma membrane, and it is accessible to antibody in the native conformation of the glycoprotein. Despite this, no neutralizing activity of 41-7 could be demonstrated in vitro. These data indicate, directly and indirectly, that this immunodominant epitope on gp41, although exposed on the viral surface, elicits antibodies lacking antiviral activity and, hence, should be avoided in future vaccine candidates.

HIV neutralizing antibodies: development and association with HIV related disease.

Serum neutralization was measured in 72 sera collected during a 5.5-year period from 10 HIV infected individuals. Neutralizing antibodies (NA) were present in all sera. NA titers ranging from greater than 40 to greater than 640 were detected in sera from 4 patients, who all remained healthy and further an increase with time of NA was observed in these 4 patients. Progression to disease was observed in 3 persons with NA titers less than or equal to 40 who also lacked or lost anti-gag antibodies. Two of these patients were HIV antigenaemic prior to development of disease, whereas antigen was not detected in the remaining 7 healthy persons. A weak positive correlation (R(S) = +0.643, p less than 0.001) was found between titers of NA and whole virus antibody (WVA), with the ratios between titers (NA titer/WVA titer) varying a 100-fold. The results suggest that the presence of NA in some cases might be related to a healthy carrier state and that a combination of low titer NA with decline of anti-gag antibodies and/or HIV antigenaemia is associated with progression to clinical disease.