In the absence of any treatment, a rapid turnover of blood virions and CD4 lymphocytes is observed in HIV-1 infection . The pathogenesis and natural history of HIV-1 infection are closely linked to the replication of the virus in vivo [2,3]. In patients with AIDS, the clinical stage of the disease is associated with the viral load, whether this is measured in the form of infectious virus, viral antigen levels in serum or viral nucleic acid content in peripheral blood mononuclear cells (PBMC) or in plasma [4,5].
Animal models such as severe combined immune deficient (SCID) mice have been used as a support for grafts of human cells [6,7]. Such humanized SCID mice were then shown to be readily infectable with HIV , and this model was recently used to test experimental gene therapy delivering interferons against HIV-1 infection .
One means of combating HIV-1 infection would be to provide seropositive patients with passive immunotherapy, using soluble molecules directed against the virus, to obtain the neutralization of HIV in vivo . Experiments involving the inoculation of virus into monkeys, together with the administration of anti-HIV-1, anti-HIV-2 and anti-SIVsm immunoglobulins, demonstrated the feasibility of the prevention of infection [11–13]. Histopathological, immunological and virological characteristics in the protected animals were strikingly similar to those observed in long-term human survivors with non-progressive HIV-1 infection .
An anti-gp41 monoclonal antibody (2F5 mAb) directed against HIV-1 displayed a potent neutralization effect in vitro and in vivo, either alone or in various combinations with other mAb or with hyperimmune globulins [15,13,16,17]. Soluble CD4 (sCD4)-mediated neutralization of SIV or HIV-1 was also demonstrated [18,19], either alone or in combination with mAb . A phase I study was successfully carried out using recombi- nant sCD4 alone  or sCD4 in combination with zidovudine .
The anti-gp41 2F5 mAb and a sCD4 -based molecule (sCD4-IgG immunoadhesin) were used in the present study. Delivering such products by gene therapy provided an efficient inhibition of HIV infection in vitro . In order to obtain constitutive secretion of 2F5  and sCD4-IgG  in vivo and to assess their antiviral properties, we developed two different genetically modified cell lines. They were incorporated in collagen fibres or synthetic tissues to form neo-organs (also designated ‘organoids') that could be grafted intraperitoneally in SCID mice. Three types of neo-organs (2F5, sCD4-IgG and 2F5 + sCD4-IgG) were generated to provide long-term delivery of recombinant molecules in vivo. These neo-organs became strongly vascularized within a few weeks of their implantation; they were not rejected and permitted secretion of the desired molecules into the blood stream of the animals. This technology has already been applied in experimental animals to treat mucopolysaccharidosis .
The questions addressed in the present study are whether the production of 2F5 antibody and sCD4-IgG immunoadhesin can be maintained continuously in vivo, and whether this production would lead to the neutralization of HIV-1 in infected humanized SCID mice.
Construction of modified cell lines
The complementary DNA [heavy chain (HC) and light chain (LC)] of 2F5 was obtained from H. Katinger (Vienna, Austria). The sequence of CD4-IgG resulted from the ligation of the leader V1/V2 segment of human CD4, the first 15 base pairs of the 2F5 hinge region and the 2F5 HC sequences. Both were inserted into retroviral vectors from Moloney murine leukemia virus, noted respectively RVTG6371 (Fig. 1a) and RVTG8338 (Fig. 1b). To avoid the gradual inactivation of retrovirally transferred expression cassettes in vivo, the promoter of mouse phosphoglycerate kinase type 1 was used. High titres of transgene products were obtained with these constructions and extinction was never observed in vitro. The dicistronic vector, RVTG6371, was used to give a sub-equivalent quantity of the two polypeptides, resulting in heavy and light chains (2F5HC and 2F5LC) of IgG3 mAb directed against the ELDKWAS linear epitope of HIV-1 gp41. The amount of sCD4-IgG obtained from RVTG8338 could not be quantified, but the molecules were detected by immunofluorescence, enzyme-linked immunosorbent assay (ELISA) and Western blot techniques.
Neo-organs were built with the genetically modified fibroblasts and a biocompatible matter made of paratetrafluoroethylene (or Gore-Tex) fibres coated with types III and I collagen threads and basic fibroblast growth factor (bFGF). The lattice of this artificial structure retracted within a few days in culture medium and the neo-organs were then ready for implantation into the peritoneal cavity of SCID mice.
Severe combined immunodeficient mice, surgical neo-organ implantation and human T cell transplantation
Ten SCID mice, controlled for agammaglobulinaemia were anaesthetized with phenobarbital. After median laparatomy, the neo-organs, maintained in bFGF-supplemented medium, were aseptically placed into the peritoneal cavity. These structures became strongly vascularized 1 or 2 weeks after their implantation in mice, as a result of the trophic and angiogenic properties of bFGF (Fig. 2a). SCID mice with neo-organs were bled weekly and checked for the presence of 2F5 mAb or sCD4-IgG in serum samples. Three weeks after the neo-organ implantation, an optimal amount of recombinant molecules was reached. At this time, 4 × 107 human CD4 cells (from the CEM T cell line) were injected intraperitoneally. Animals receiving CEM cells in the absence of previously implanted neo-organs were used as controls.
Enzyme-linked immunosorbent assay of 2F5 monoclonal antibody and the soluble CD4-IgG molecule
Measures of 2F5 or sCD4-IgG in vitro and ex vivo were performed in cell culture supernatants and in plasma, respectively, using ELISA assays. Briefly, microplates were coated by overnight incubation with purified ELDKWAS peptide or with gp160 proteins of the HIV-1 envelope. Inactivated plasma from neo-organ-implanted SCID mice from each group were then tested. 2F5 mAb or sCD4-IgG were detected using horseradish peroxydase-conjugated goat anti-human IgG. The coloured reaction was revealed using orthophenylenediamine as the substrate and stopped 3 min later by the addition of sulphuric acid 6 M. Optical densities were determined at 490 nm with the ELISA reader.
Immunofluorescence detection of 2F5 monoclonal antibody
Adherent cells were fixed for 20 min in methanol acetone (v/v) before being treated. One step of incubation with a fluorescein-isothiocyanate-conjugated goat antibody directed against human IgG (heavy and light chains) diluted at 1 : 200 in phosphate-buffered saline × 1 with 5% fetal calf serum was performed. Ethidium bromide 1 : 100 in phosphate-buffered saline × 1 was used to colour the nuclei.
HIV-1 challenge in vivo
Four weeks after the intraperitoneal injection of CEM cells, SCID mice were challenged intravenously with 1000 TCID50 of HIV-1 (Lai or MN). The virus titration was carried out in CEM cell cultures, using the Nara technique .
Measures of plasma and cellular viral loads, and reverse transcriptase activity
On one part of the cells removed from the spleen, the cellular proviral DNA was analysed, using a quantitative–competitive polymerase chain reaction (PCR). The total DNA was extracted according to the tri-reagent method (Euromedex, Strasbourg, France) from HIV-1-infected and from non-infected CEM cells harvested from the spleens of killed SCID mice. The plasma viral load was measured using the NASBA kit. A 140 base pair fragment of the gag gene was amplified from 2 μg DNA by quantitative–competitive PCR (HIV-1 PCR MIMIC Quantitation System kit; Clontech, Cambridge Bioscience, Cambridge, UK) using a SK 462/SK 431 primer pair in the presence of a 260 base pair heterologous fragment. A 10-fold dilution of the competitor (106 : 1 copies) allowed the determination of the equimolarity of gag in each sample. The analysis of PCR products was performed by Southern blot hybridization using 32P-labelled SK 102 and MIMIC probes (Clontech).
On the other part of the cells removed from the spleen, liver and tumour, cultures were performed to measure reverse transcriptase activity .
NIH-3T3 fibroblasts were stably transduced with RVTG6371 or RVTG8338 encoding mAb 2F5 and sCD4-IgG, respectively (NIH-3T3TG6371 or NIH3T3TG8338). The resulting cells were shown constitutively to produce the mAb or the immunoadhesin through the analysis of culture supernatants by a Western blot assay and ELISA (data not shown) and by a specific immunofluorescence technique (Fig. 1c).
Three groups of experiments were carried out: 2F5, sCD4-IgG or 2F5 plus sCD4-IgG neo-organs were built and implanted into SCID mice as described in the Methods section.
In-vivo release of 2F5 and sCD4-IgG from genetically modified tissues
In neo-organ implanted SCID mice, the follow-up of 2F5 production showed an increased secretion during the first 5 weeks after grafting (Fig. 2b). The mean plasma levels of 2F5 increased from 167 to 1000 ng/ml between weeks 1 and 5 (Lai-inoculated group), and from 114 to 2000 ng/ml between the same weeks (MN-inoculated group). At week 6, means of 450 and 1325 ng/ml 2F5 were found in the plasma of mice of the Lai and MN groups, respectively.
We did not have any purified sCD4-IgG standard to quantify the production of the immunoadhesin in vitro and in vivo. Nevertheless, we were able to detect the molecules in cell supernatants and in mouse plasma by a qualitative ELISA assay as described in the Methods section (data not shown).
In-vivo decrease of plasma viral load induced by 2F5 monoclonal antibody
As shown in Table 1, seven control SCID mice (four Lai and three MN) and six SCID mice with 2F5 mAb-producing neo-organs (four Lai and two MN) were tested for plasma viral load.
In control animals, the plasma viral load increased significantly from day 5 to day 12 after HIV-1 challenge. The number of RNA copies reached 29–120 × 107 and 82–130 × 107 for Lai and MN isolates, respectively.
In mice implanted with 2F5-producing neo-organs, the plasma viral load was approximately 100 000-fold lower than that in control mice at day 12 (Table 1) both in the Lai and the MN groups. All tested implanted mice had a number of RNA copies below 16.5 × 103 at day 12.
In-vivo decrease of cellular viral load by 2F5 monoclonal antibody
At the time of death, quantitative detection of HIV-1 proviruses was performed by PCR in one part of the spleen cells recovered from control and from 2F5 neo-organ-implanted SCID mice inoculated with HIV-1. Proviral DNA was detected in all samples, but at different levels (Fig. 3). It was approximately 100-fold lower in cells from mice with a 2F5 neo-organ than in cells from control mice. Whereas HIV-1 (Lai) experimental inoculation resulted in infection with either 3 × 106 or 3 × 105 HIV-1 copies in the five control mice, in SCID mice grafted with a 2F5 neo-organ, 3 × 105 HIV-1 copies, 3 × 104 HIV-1 copies or 3 × 103 HIV-1 copies were detected. In mice injected with the MN strain, no statistically significant difference in the detection of intracellular HIV-1 provirus was observed between controls and 2F5-producer animals (data not shown).
Cell cultures were performed using human T cells (CEM cells) recovered from the second part of various organs (spleen, tumour and liver) of grafted SCID mice. When CEM cells were removed from spleens, livers and tumours of mice implanted with 2F5 neo-organs, very low reverse transcriptase activity was consistently observed, in comparison with cultures obtained from HIV-infected control SCID (data not shown).
In-vivo decrease of plasma viral load by soluble CD4-IgG and by combined 2F5 plus soluble CD4-IgG
A separate experiment to compare 2F5 mAb with sCD4-IgG showed that both compounds had comparable inhibitory effects on the plasma viral load of HIV Lai- or HIV MN-infected SCID–CEM mice (Table 2). As we used a different stock of Lai virus than in the previous experiment (Table 1), the mean plasma viral load was higher. However it was significantly lower in all groups of implanted mice than in controls.
The effect of sCD4-IgG and 2F5 were therefore of the same order of magnitude. When both molecules were produced together in vivo, again an important anti-HIV activity was demonstrable (Table 2). Whether or not a synergism might exist is difficult to state from our data because there are no statistically significant differences between the three groups of treated animals. At day 10, admittedly the viral load tended to be lower in the group with two molecules but, even then, the reduction did not reach a significant difference with either sCD4-IgG or 2F5 alone.
In-vivo decrease of cellular viral load by soluble CD4-IgG and combined 2F5 plus soluble CD4-IgG
At the time of death, the number of HIV-1 proviral DNA copies was low in the spleen cells of each group of mice implanted with neo-organs secreting 2F5, sCD4-IgG or 2F5 plus sCD4-IgG (Table 3) compared with controls. In the last group, a more homogeneous and potent effect was observed.
Our experiments in humanized SCID mice demonstrate that the continuous production of either neutralizing anti-HIV antibodies or sCD4-IgG can greatly reduce the HIV-1 load in vivo.
Several hybridomas, prepared using cells from HIV-1-infected individuals, have been established and produce human mAb against various epitopes of HIV-1 . With the aim of clinical use, their in-vitro evaluation has been carried out, especially using syncytium inhibition, neutralization, antibody-dependent cell cytotoxicity and complement-dependent cytotoxicity. The 2F5 mAb was shown to be the most effective in vitro [24,29]; it recognizes a highly conserved continuous epitope on the transmembrane envelope gp41 molecule of HIV-1 [30,31]. A dose–effect relationship was demonstrated in vitro between the quantity of antibody and the degree of virus neutralization. Administered to patients in vivo, 2F5 was more efficient in reducing the HIV load than the endogeneously produced antibodies resulting from the presently available vaccinations (Katinger H, personal communication). A potent passive immunoprophylaxis could be envisaged using various mAb including 2F5 (anti-gp41), 694/98D (anti-V3 loop), 2G12 (anti-gp120) and F105 (anti-CD4 cell-binding site) in quadruple combinations . The transient effects of the infusion of a variety of anti-HIV antibodies have been studied in humans  and in animals, especially in chimpanzees  and in SCID mice reconstituted with human peripheral blood leukocytes [34,35]. All results obtained demonstrated that a potent neutralizing activity against a broad range of divergent HIV-1 variants and primary isolates in vitro and in vivo [1,14,31,36] is closely related to the levels and the frequency of 2F5 administration. These data suggested that a more potent and prolonged effect might result from an in-vivo delivery of 2F5 by a bioengineering procedure such as that accomplished by gene therapy. In order to assess the efficiency of the sustained secretion of antiviral molecules in vivo, we used neo-organ technology . Our experiments were based on NIH3T3 murine fibroblasts genetically modified in vitro. Their implantation into SCID mice led to tumour development, which limited long-term studies. The continuous production of 2F5 could, however, be obtained for up to 6 weeks at serum concentrations close to 1 μg/ml.
Similarly, a soluble form of the CD4 molecule has been shown to block the interaction of CD4 cells with gp120, thus inhibiting HIV infection [37,38]. It was further demonstrated that the effect was dose-dependent when sCD4 cells were maintained throughout cell culture . The continuous secretion of sCD4 by somatic transgenesis ensured their presence for 2 months after a single transgenesis in mice, whereas administration in vivo resulted in the short-term presence of the molecule, for a few hours . A second-generation CD4 -based molecule involved the genetic coupling of a portion of the CD4 structure to the Fc fragment of the IgG molecule. Such immunoadhesins  displayed the same affinity as sCD4 for gp120 and have a longer half-life in vivo . Other systems using sCD4 have been tested in vitro : for instance CD4-Pseudomonas exotoxin (CD4-PE) induced a delay in, but not a genuine inhibition of, HIV replication in human peripheral blood lymphocytes .
In previous pilot experiments, we confirmed the antiviral efficacy of the recombinant molecules. Using 2F5 neo-organs, we observed that the intensity of the antiviral effect was dependent on the dose of antibody produced in vivo (data not shown). It was found that plasma levels of 2F5 approximating 1 μg/ml in SCID mice were required to induce a regular and significant reduction in the virus load. In the present experiments, with the endogeneous production of 2F5 after neo-organ implantation, not only was the number of RNA copies of HIV-1 in SCID–CEM mice very significantly decreased, but also the cellular viral load and reverse transcriptase activity were reduced.
When produced by neo-organs in vivo, we observed that the sCD4-IgG molecule was effective in significantly reducing the viral load of HIV-infected SCID–CEM mice.
Either 2F5 or sCD4-IgG could inhibit HIV infection in vivo. Because of their high affinity for highly conserved epitopes of the gp120 and gp41 glycoproteins of HIV-1 envelope, the CD4-IgG and 2F5 molecules display a broad spectrum of action [11,25]. Both were shown to recognize either laboratory or primary HIV isolates [15,19].Their potential antiviral efficiency was directly related to their ability to interact with the HIV-1 envelope. This would affect HIV-1 propagation by preventing the virus from infecting its target cells because of their competitive or neutralizing properties. When the two molecules were produced together, an additive effect occurred in several mice. The soluble CD4 molecule would facilitate the action of neutralizing antibodies such as 2F5 because of the conformational change of the HIV-1 envelope induced by its interaction with gp120. Actually, the specific epitopes recognized by the antibodies would be more accessible. This latter activity can be interpreted as the result of CD4 binding to the gp120 of HIV, then larger exposure of gp41, and finally increased binding of 2F5 to gp41. It is known that the CD4 molecule induces conformational changes in gp120, permitting access to gp41 [44–46]. In any case, anti-gp41 antibodies cannot be regarded as being responsible for a full and direct neutralizing activity because the target molecule becomes accessible only after virus attachment to the CD4 target cell . The use of a sCD4 molecule could thus help in gaining access to gp41 epitopes. A more synergistic effect might be obtained in animal models in which not only human CD4 cells are present in vivo, but also all the other components of the human immunological network.
Our data showed the efficacy of the continuous delivery of soluble neutralizing molecules in vivo against both the Lai and MN isolates of HIV-1, suggesting the possibility of gene therapy. Ideally, the treatment should be targeted not only at cell-bound HIV but also at free virions. The addition of sCD4-IgG to 2F5 may contribute to the accessibility of free virions. It is also likely to increase the efficacy of 2F5 in the inhibition of HIV transfer between cells, such as that demonstrated from dendritic cells to T cells .
The above-described gene therapy approach could complement the presently available anti-HIV therapies to treat HIV-infected patients even more efficiently and to prevent maternal transmission of the virus to the fetus or the neonate. The use of neo-organs in therapeutics is quite limited by safety problems. Established cell lines induce tumour formation in vivo, and genetically modified cells can escape from the neo-organ leading to the dissemination of cells and transgenes. The 3T3 murine fibroblasts induce tumours in immunodeficient mice and cannot be considered for human use. Modification of the techniques will be required for preclinical studies. The use of a direct gene therapy based on the systemic administration of adenoviral vectors in the presence of HIV-1 primary isolates is currently under investigation.
The authors would like to thank Professor H. Katinger from Vienna for providing the 2F5 cDNA. They are also grateful to P. Leroy, R. Sembeil and M. Fardeheb for useful help in data analysis.
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