Expression of HIV-1 envelope gene by recombinant avipox viruses

Expression of HIV-1 envelope gene by recombinant avipox viruses A n t o n i a Radaelli** a n d C a r l o D e G i u l i M o r g h e n * ~

Recombinant canarypox (CP) and fowlpox (FP) viruses that contained two forms o f the HIV-1 (SF2 strain) env 9ene were engineered and their expression analysed in chick, simian and human cells. These vectors can efficiently replicate in avian but not in mammalian cells, in which infection is abortive. The two forms, consistin9 o f the entire env open reading frame (IS +) or o f the same 9ene lackin9 the putative immunosuppressive ( I S - ) region (amino acids 583-599), were individually inserted into the two virus vector backgrounds. In order to avoid premature transcription termination of the foreign 9ene and to improve protein expression, a mutagenesis was also performed within the T 5 N T motif without alterin9 the amino acid sequence. By immunoprecipitation analyses, cells infected with CP and FP recombinants expressed HIV-1 env polypeptides of the appropriate molecular weight. We observed that the 91)160 precursor was proteolytically cleaved except in MRC-5 cells infected with the I S - recombinants and that these polypeptides were 9lycosylated. Further analysis o f these recombinant viruses by indirect immunofluorescence and syncytia inhibition assays indicated that the 9p120/gp41 complex was present on the surface of infected cells, the number of syncytia bein9 significantly lower when cells were infected by the C P I S - or F P I S - recombinants. Moreover, sera o f immunized rabbits revealed the presence oJspecific antibodies in animals inoculated either with CP or with FP recombinants. These new constructs, which are unable to support a productive infection in human cells', might therefore also be a 9ood anti-HIV-1 candidate vaccine in seropositive hosts. Keywords:Human immunodeficiency virus type 1; recombinant avipox virus; expression;

The env gene of the human immunodeficiency virus type 1 (HIV-1) encodes a precursor glycosylated protein which is proteolytically cleaved inside the cell into the two glycoproteins gp 120 and gp411. The external subunit, gp 120, is responsible for initiating the infection by binding of its C-terminal region to the CD4 molecule 2, which is the main viral receptor site 3, although functional studies also demonstrate CD4-independent infection by HIV-14 or the requirement for other molecules 5. The gp41 transmembrane subunit mediates fusion of the viral and host cell membranes 6, thus allowing viral entry. Although neutralizing antibodies have also been identified against the core proteins v, the H1V-1 epitopes that reside within env gene products are more important as targets for immune attack, as they are essential for early steps in virus-cell interaction 8'9. Nevertheless, only some of them can elicit neutralizing antibodies and, even then, *Institute of Pharmacological Sciences, tC.N.R. Center for Cytopharmacology, and tDepartment of Pharmacology, University of Milano, Italy. °°To whom correspondence should be addressed at: Department of Pharmacology, University of Milano, Via Vanvitelli 32, 20129 Milano, Italy. (Received 16 June 1993; revised 25 November 1993; accepted 30 December 1993) 0264-410X/94/12/1101-09 ~ 1994 Butterworth-Heinemann Ltd

env

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the immune response may be unable to prevent HIV- 1 infection after challenge10 A high immunodominant epitope was also identified on the N-terminus of gp41, which is responsible for humoral immunity in healthy seropositive individuals 11 and specifically inhibits lymphoproliferation in vitro 12. This region shows a sequence homology to the gp21E transmembrane protein of human T-lymphotropic viruses types I and II (HTLV-I and -II) and to an immunosuppressive (IS) region of the pl5E of murine (MuLV) and feline (FeLV) retroviruses 13 More recent data 14 demonstrated that human monoclonal antibodies (mAbs), directed against gp41 immunodominant epitopes including the above-mentioned IS region, are responsible for antibody-dependent enhancement (ADE) of HIV-1 infection in vitro. This phenomenon, already described by several authors ls'16 and requiring both the CD4 and the FcyR receptors 5, might be involved in the lack of protection of immunized animals. In this report, we present four new recombinant avipox viruses expressing the env gene of the SF2 strain of HIV-1, in two of which the putative IS region is deleted. The canarypox and fowlpox viruses, recently developed for the expression of foreign genes 17'1s, may represent

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innovative and safer vaccines for immunocompromised hosts, since their productive replication is restricted to avian species 19.

MATERIALS AND METHODS

Cells Specific pathogen-free (SPF) primary chick embryo fibroblasts (CEF), monkey kidney cells (Vero) and the human cell line MRC-5 were cultivated in Eagle's minimum essential medium (MEM) containing 10% fetal bovine serum (FBS) (Gibco) and supplemented with antibiotics. Human CD4-positive T-cell lymphocytes, HuT 78, were kindly provided by Dr G. Giraldo, I.N.T. (Naples, Italy) and HuT 78/HIV-lsF 2 (E-line) cells by NIAID AIDS Research and Reference Reagent Program (Rockville, MD). They were both cultured in RPMI 1640 medium with 15% FBS.

Viruses The canarypox (CP) virus, obtained from Institut M6rieux (Lyon, France), was used for making recombinants vCP60 and vCP61. The fowlpox (FP) virus, obtained from the same source, was used for making recombinants vFP62 and vFP63. All the viruses were grown on chick cells and used to infect Vero, MRC-5 and CEF cells.

Construction of plasmids The lambda clone containing the entire HIV-ls~ 2 genome was kindly provided by Dr J. Levy (San Francisco, CA) and has been described by SanchezPescador et aL 2°. Plasmid pMP7MX373, subcloned in pUC13 and containing the env sequences from - 1 relative to the initiation codon (ATG) for the env gene product to 837 bp downstream of the termination codon (TAA), was kindly provided by Dr M. Perkus (Virogenetics, Troy, NY). These env sequences were excised from pMP7MX373 by digestion with E c o R I and HindIII and inserted into the plasmid vector pIBI25 (IBI, New Haven, CT) where in vitro mutagenesis reactions 21 were performed. First, using oligonucleotide 5'-AGA-GGG-GAA-TI'C-TTC-TAC-TGC-AAT-ACA-3', we generated a T ~ C transition to disrupt the T5CT motif at nucleotide 5' position 6928 6934 of the SF2 genome. This mutation does not alter the amino acid sequence of the env gene and creates an E c o R I site, which was used to screen for mutagenized plasmid clones. Two other mutageneses were performed to remove the sequence coding for the n e f regulatory protein and the LTR sequence (LTR region) from the 3' end of the gene and to delete the putative IS region (amino acids 583 to 599: Leu-Gln-Ala-Arg-Val-Leu-Ala-Val-GluArg-Tyr-Leu-Arg-Asp-Gln-Gln-Leu) 1~, using oligonucleotide 5'-TTG-GAA-AGG-CTT-TTG-CTA-TAA-AAGCTT-GCA-TGC-CAC-GCG-TC-3' for the former and oligonucleotide 5'-ACA-GTC-TGG-GGC-ATC-AAGCAG-CTA-GGG-ATT-TGG-GGT-TGC-TCT-3' for for the latter. Mutagenized clones were identified by hybridization and restriction analysis. The sequence was confirmed using the dideoxynucleotide chain termination method 22. The clones mutagenized so that both the IS and the LTR regions were deleted or with only the LTR region deleted were designated p I B I 2 5 m u t 3 e n v 4 0 and

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pIBI25mut2env22, respectively. A 2.5 kb S a m I / H & d l l I fragment containing the entire env gene was derived from p I B I 2 5 m u t 3 e n v 4 0 and from p I B I 2 5 m u t 2 e n v 2 2 and inserted into pCPCV1 and pFPCV2 insertion plasmids digested with S m a I / H i n d I I I . pCPCV1 and pFPCV2 are insertion vectors that allow for the generation of CP and FP recombinants, harbouring the foreign genes in loci C3 and F7 respectively. Both plasmids, kindly provided by Dr J. Tartaglia (Virogenetics, Troy, NY) contain pUC sequences, the vaccinia virus H6 promoter and a multiple cloning region juxtaposed 3' to the regulatory element. Oligonucleotide 5'-CCG-TTA-AGT-TTG-TAT-CGTAAT-GAA-AGT-GAA-GGG-GAC-CAG-G-3' was used for in vitro mutagenesis reactions according to Mandecki's method 23 to make a precise ATG:ATG construction with the VVH6 promoter 24 and the env sequences. Potential mutants were screened for the loss of the S m a I site. Plasmid clones devoid of a S m a I site were identified and confirmed by nucleotide sequence analysis. Properly mutagenized plasmid clones were identified and designated as p C P e n v I S + or p C P e n v I S and p F P e n v l S + or p F P e n v l S .

In vivo recombination and purification of recombinants In vivo recombination was performed by introducing plasmid DNA into infected cells by calcium phosphate precipitation both for CP and for FP recombinants, as described by Panicali and Paoletti25. Plasmids pCPenvIS + and p C P e n v I S - were used to make recombinants vCP61 and vCP60 respectively, while plasmids p F P e n v I S + and pFPenvIS were used for recombinants vFP63 and vFP62. For convenience, the two CP recombinant viruses will be referred to as CPIS + and CPIS- and the two FP recombinants as FPIS + and FPIS- respectively. Recombinant plaques were selected by autoradiography after hybridization with a 32p-labelled env probe and passaged serially several times t o ensure purity, as previously described 2('.

Radioimmunoprecipitation analysis (RIPA) Cells were infected with 10 plaque forming units (p.f.u.)/cell in modified Eagle's methionine-free medium (MEM met-). After 2 h, 20/iCi ml-1 of 35S-methionine were added, using the same medium supplemented with 2% dialysed FBS (Flow). Sixteen hours postinfection, cells in 2 ml of medium were harvested by resuspending in 1 ml lysis buffer (150ram NaC1, 1 mM EDTA, 10ram Tris-HC1, pH 7.4, 0.2 mg ml- 1 PMSF, 1% NP40, 0.01% sodium azide) and 0.6 TIU aprotinin (Sigma) per Petri dish, scraped into Eppendorf tubes, and the lysate was clarified by spinning at 9000g for 20 rain at 4°C. Immunoprecipitation was performed as already described 17 either with 2/A pooled and preadsorbed human HIV-Iseropositive serum, previously heat-inactivated, or with commercial (DuPont) anti-gpl20 and anti-gp41 monoclonal antibodies (mAbs), or with goat HIV-1 SF2-specific anti-gp120 atibodies, kindly provided by NIAID AIDS Research and Reference Reagent Program (Roekville, MD). Proteins were resolved by 8% SDS- polyacrylamide gel electrophoresis (PAGE) and fluorographed. The same procedure was utilized in deglycosylation experiments where the immunoprecipitates were incubated with 0.01 units of endoglycosidase F/N-glycosidase F (Endo F) (Boehringer Mannheim) in the presence of 0.025% SDS, as described by Matthews et al. 27.

HIV-1 env gene expression by avipox viruses: A, Radaelli and C. De Giuli Morghen

Immunofluorescence Cells were seeded at a density of 5 x l0 s per 35 mm 2 dish on sterile glass coverslips and, after 24 h, infected with either the wild-type or the recombinant viruses at 10 p.f.u./cell. After overnight growth, cells were rinsed twice with phosphate-buffered saline (PBS) containing 0.2% bovine serum albumin (BSA) and 0.1% sodium azide. For cytoplasmic immunofluoreseence (IF), cells were washed in PBS-BSA for 5min, fixed with 100% cold acetone for 5rain at - 2 0 ° C and washed again. For membrane IF, cells were either fixed for 20 min with fresh 2% paraformaldehyde in calcium- and magnesium-free PBS (PBS-) or directly incubated with the specific antibodies. The reaction was performed for 1 h at room temperature with polyclonal goat anti-HIV-lsv2 gpl20 or human HIV-l-positive sera diluted 1:500 in PBS-BSA buffer as well as with commercial (DuPont) anti-gpl20 and anti-gp41 mAbs diluted 1:200 in the same buffer. All subsequent steps were performed as already described 17. Syncytia inhibition assay Confluent Vero cells were infected at 10 p.f.u./cell for 1 h at 37°C either with the CPIS +, C P I S - , FPIS + or F P I S - recombinant viruses or with the CPwt or FPwt counterparts. Mock-infected cells served as a control. After overnight incubation, the medium was removed and CD4 + H u T 78 lymphocytes, resuspended in R P M I with 7.5% FBS, were added at a ratio of 5:1 to Vero cells. Alternatively, Vero cells were treated with 5/~g ml 1 recombinant soluble CD4, kindly provided by Dr J. Mous (Hoffmann-La Roche Ltd, Basel, Switzerland), in 1 ml RPMI and, after 1 h incubation, H u T 78 cells were added. Cell fusions were examined by an inverted phase-contrast microscope (Leitz Diavert) 20 and 42 h after infection. Immunization of animals New Zealand White 2-month-old rabbits (Charles River, Italy) were inoculated with 1 x 10 8 p.f.u, of sucrose gradient-purified recombinant or wild-type viruses by multiple intradermal injections on the upper back after depilation. Booster immunizations were repeated five times, the first 1 month after priming and then at 15-day intervals. The last booster was given with a lower viral dose of 1 x 1 0 7 p.f.u. Bleeding was performed from the central artery of the ear the day before inoculation, the serum fraction was isolated, and aliquots were frozen at _ 40°C. Enzymed-linked immunosorbent assay (ELISA) Rabbit

sera

were

tested

for

the

presence

of

anti-env-specific antibodies by using an ELISA performed

essentially as already described 28 except that sonicated E-line cells in 0.05 M carbonate-bicarbonate buffer, pH 9.6, were plated in 96-well microtitre plates. The absorbance (A) of each well was read at 492 nm with a Titertek Multiskan (Flow) spectrophotometer. Experimental A values greater than three times the mean A value obtained with negative controls were considered as the positive-reactivity cutoff level. Both preimmune rabbit sera and sera from wild-type animals were used as negative controls, whereas anti-HIV-lnm hyperimmune

rabbit serum, kindly provided by Drs Cattaneo and Achilli (Policlinico San Matteo, Pavia, Italy), was used at 1:1000 dilution as a positive control. The endpoint antibody titre of sera was considered as the highest dilution resulting in an A value greater than the positive cutoff. RESULTS Construction of the recombinant viruses Four different recombinant viruses were prepared with the env gene of the HIV-lsv 2 isolate inserted downstream from a vaccinia transcriptional early-late promoter, designated VVH6. All the plasmids were constructed so that the ATG initiation codon would immediately follow the VVH6 promoter in the recombinant viruses. The region following the TAA termination codon was also removed to obtain a precise construction. Moreover, since a vaccinia virus transcriptional termination signal 29 was present towards the Y-terminus of the gp 120-coding gene, a mutagenesis was performed to allow the uninterrupted transcription of the whole heterologous env gene. Both C P I S - and F P I S - recombinants were obtained by deletion of a 51 bp region, corresponding to amino acids 583-599, located near the 5'-terminus of the gene coding for the gp41 protein. This region is partially homologous to a corresponding murine, feline and human retrovirus sequence 13 and has been shown to be immunosuppressive in vitro 11,12. It was therefore designated as the IS region. Expression and processing of the HIV-I

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As shown by radioimmunoprecipitation analysis of the three different infected cell lines, Vero, MRC-5 and CEF, metabolically labelled with 35S-methionine, all four recombinants directed the expression of the gpl60 envelope precursor, although at different levels (Figures 1A and B). In particular, after infection with CPIS + and C P I S - recombinants (Figure 1A), the gpl60 protein was normally synthesized and processed into the gpl20 protein, the only exception being the MRC-5 cells infected by C P I S - where cleavage of the gpl60 precursor to gpl20 did not seem to take place. Gp41 was undetectable in all cells. The results were analogous in F P recombinants (Figure lB). Both CPIS + and FPIS + recombinants revealed a higher expression of HIV-1 env proteins than their IS- counterparts. A protein with an apparent molecular weight of 4 3 k D a was also precipitated for all samples, the amount being higher when higher amounts of the env gene products were coprecipitated. No specific proteins were observed when cells were mock-infected or infected by wild-type viruses. Moreover, no env proteins from the cell lysates could be detected when immunoprecipitation was performed by a commercial (DuPont) anti-gpl20 mAb. In contrast, the SF2specific anti-gpl20 goat polyclonal antiserum was able to precipitate both gpl60 and gpl20. The anti-gp41 mAb could precipitate not only gp41 and its precursor gpl60, but also a glycoprotein with an apparent molecular mass of 120 kDa, although differently in the various cell lines (data not shown). Therefore, to confirm that a normal process of glycosylation of the env gene products had occurred and that the gpl20 band was the real product of the

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HIV-1 e n v gene expression by avipox viruses: A. Radaelli and C. De Giuli Morghen VERO

MRC-£

Conversely, almost 100% of MRC-5 cells, which are also restrictive (Figures3d-f), were positive, although the immunofluorescence intensity was lower. Replicationpermissive CEF cells (Figures3g i) always showed the presence of the antigen in almost all the cells. External IF was observed on samples prefixed with paraformaldehyde before adding the first antibody, but only when using polyclonal sera (Figures3l-n) or anti-gp41 mAb (data not shown). However, even after paraformaldehyde fixation, a slight intracellular IF was observed in some cells infected by the IS + and IS recombinants, probably due to the cytotoxic and permeabilizing effect of the viral infection per se. No IF was detected in cells infected by wild-type parental CP or FP viruses or by using various other anti-gp41 or anti-gp120 mAbs.

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Evidence that the e n v gene product is functionally expressed by the CP and FP recombinants Although the presence of the gp41 fusion-mediating protein could not always be detected by radioimmunoVERO

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Figure 1 Immunoprecipitation of HIV-1 envelope glcoproteins from lysates of ceils infected with canarypox (A) and fowipox (B) recombinants. Labelled proteins from lysates of Vero, MRC-5 and CEF cells were immunoprecipitated wih pooled preadsorbed human HIV-l-seropositive specific sera and resolved on an 8% SDS-PAGE gel. The different cell lines and the inoculum used for infection are indicated at the top of the figure. The lanes marked as mock were loaded with proteins from non-infected cells. Molecular weight markers are indicated

NH2-terminal portion of the env gene and not only a trimer of the COOH-terminal part (gp41), and to rule out the possibility that gpl60 was simply an oligomeric form of gp413o,31, deglycosylation of immunoprecipitates was performed using Endo F. This experiment would also confirm the presence of carbohydrate-related structures, which have been demonstrated to be important targets for virus neutralization 3a. As expected, the deglycosylated products of the immunoprecipitates (Figure 2) revealed the presence of two polypeptides with apparent molecular masses of 97 kDa and 55 kDa, corresponding to the unglycosylated gpl60 and gpl20 env gene products respectively 1v'33, thus confirming the regular processing of the two glycoproteins. A different expression pattern was observed for the different cell lines infected by the IS + and ISrecombinants in the immunofluorescence assay. In Vero cells (Figures 3a-c), in which the avipox viruses do not replicate, the recombinants showed a high-intensity cytoplasmic immunofluorescence in 30-50% of the cells.

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Figure 2 Immunoprecipitation of HIV-1 envelope glycoproteins from lysates of cells infected with canarypox (A) and fowlpox (B) recombinants. Proteins are shown before (a) and after (b) deglycosylation with Endo F. Immunoprecipitates were prepared as described in Figure 1. The molecular weights of unglycosylated proteins are indicated on the right

HIV-1 env gene expression by avipox viruses: A. Radaelli and C. De Giuli Morghen a

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Indirect immunofluorescence of Vero (first and fourth row), MRC-5 (second row) and CEF cells (third row) infected with CPwt (a, d, g, I), CPIS + (b, e, h, m) and CPIS (c, f, i, n). Cells were fixed either with 100% acetone (first, second and third rows) or with 2% paraformaldehyde (fourth row), and incubated with human HIV-l-positive serum followed by FITC-conjugated anti-human IgG

precipitation analysis, extensive cell-to-cell fusion was observed when Vero cells, infected with the recombinant viruses, were overlaid by CD4-positive H u T 78 cells (Figure 4). The number of fused cells 20 h after infection did not increase I day later, and was significantly higher, as evidenced by the larger size of syncytia, in cells inoculated with CPIS + or FPIS + (Figures4e and 4b) than in cells inoculated with C P I S - or F P I S recombinant viruses (Figures 4f and 4c). No cell fusion could be observed in mock-infected cells or in cells infected with the wild-type viruses (Figures 4a and 4d). To confirm that fusion between the Vero cells infected with the recombinant poxviruses and the H u T 78 cells was due only to the expression of gp120 and gp41 env-specific proteins, different concentrations of soluble CD4 were tested to see whether fusion could be competitively blocked. It was found that 5/~g m1-1 of soluble recombinant CD4 protein completely inhibited fusion of H u T 78 cells by Vero cells infected either by the C P I S + ' a n d C P I S - or FPIS + and F P I S recombinants (data not shown).

HIV-l-specific humoral response is elicited in rabbits immunized by CP or FP recombinants Sera from immunized rabbits were tested in an ELISA assay to determine the presence of antibodies reactive with the HIV-1 env proteins, expressed by E-line cells, adsorbed to a 96-well plate. Preimmune serum from the same rabbit was used as a negative control. In all the vaccinated rabbits an immune response was elicited against their respective poxvirus vectors (data not shown). One month after priming (Figure5), all the animals injected with the recombinants showed seroconversion. The most significant increase in antibody titre was generally observed after the first or second booster. DISCUSSION Recombinant vaccinia viruses have already been designed either expressing the env gene products 34-37 or the core proteins 38-41 and they have been tested as immunogens

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Figure 4 Syncytia formation induced by Vero cells expressing HIV-1 envelope glycoproteins on CD4 + HuT 78 lymphocytes. After incubation, infected Vero cells were overlaid with HuT 78 cells and cell fusions were examined 20 h after infection. First row: (a) FPwt-, (b) FPIS ÷-, or (c) FPIS-- infected cells. Second row: (d) CPwt-, (e) CPIS +-, or (f) CPIS - infected cells

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BLEEDINGTIME(WEEKS) Figure 5 HIV-1 specific antibody titres in rabbits immunized with CPIS + and CPIS- ((A) animal nos. (m) 21, (@) 22 ( x ) 23, and (*) 24) or FPIS ~ and FPIS- ((B) animal nos. ( . ) 27, (@) 28 ( x ) 50, and (*) 53) recombinants. Rabbits were bled after 4, 6, 8, 10 or 12 weeks, as indicated. Ninety-six-well microtitre plates were covered with disrupted HIV-lsF2-producing human cells. The bound antibodies were revealed by horseradish peroxidase-conjugated anti-rabbit IgG and o-phenylenediamine substrate as described in Materials and methods. Experimental A values greater than three times the mean A value obtained with negative controls were considered as the positive-reactivity cutoff level. The endpoint antibody titre of sera was the highest dilution resu Iti ng in an A value greater than the positive cutoff. Each point of the graph represents the average of duplicate assays

mainly in chimpanzees 42, but also in macaques 43, mice 44 and humans 45. These vaccines were able to induce a humoral response and could sometimes raise a T-cell mediated immunity, but sera from the inoculated animals were not always able to neutralize HIV-1 infection in vitro. Furthermore, passive transfer of high-titre neutralizing antibodies failed to protect chimpanzees from challenge with HIV-146, although a chimeric m o u s e h u m a n m A b could prevent infection with a homologous H I V - I strain 47.

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In this paper, we describe the construction of new poxvirus recombinants using C P and F P virus vectors in which the env gene from the SF2 isolate of HIV-1 was inserted under the transcriptional control of a vaccinia promoter. F P and CP viruses have recently been developed as live vectors, as they can trigger a protective immune response although unable to start a productive infection in cells derived from non-avian species 17'48. The availability of these recombinant vectors, which are unable to replicate in mammalian cells,

HIV-1 env gene expression by avipox viruses: A. Radaelli and C. De Giuli Morghen

could also prove extremely advantageous for vaccination of subjects previously inoculated with vaccinia virus 49. In addition, 51 nucleotides within the gp41 domain of the HIV-1 e n v gene were deleted in both FP and CP recombinants to verify the effects of elimination of such a putatively immunosuppressive sequence. Furthermore, in order to improve foreign protein expression and possibly the subsequent immune response, we produced a transition within a TTTTTCT early transcription termination signal inside the env gpl20 coding region, which has recently been demonstrated to cause premature termination of transcripts 29. Only one T5NT motif was found in the env gene of HIV-lsF 2 in comparison to the two T5NT sequences of the HIV-111m isolate. The present study demonstrates that efficient expression and processing of HIV-1 e n v gene proteins can be achieved by recombinant CP and FP vectors. By immunoprecipitation assay, no significant difference was observed in expression of the gpl60 precursor in the three different cell lines, Vero, MRC-5 and CEF. Therefore, although chick cells are the natural and permissive host of these viruses, antigens can also be expressed and processed by primate and human cells, except that the gpl60 product of the IS- recombinant was not cleaved by MRC-5 cells. The failure to detect the presence of gp41 may be explained by posttranslational assembly into trimers and tetramers as well as into other oligomeric forms 3°'5°, which comigrate with gpl20 and gpl60 bands 31. In fact, an anti-gp41 mAb revealed the presence not only of gp41 and its gpl60 precursor, but also of a band of 120 kDa (data not shown). These results suggest that, although this protein is not detectable in immunoprecipitation assays by polyclonal antibodies, it is mainly present as an oligomer. Deglycosylation experiments demonstrate that the avipox virus expression system can drive the synthesis of glycosylated env gene products. This would allow the generation of conformation-dependent immunoglobulins, which have been proved to be essential for the binding of some neutralizing antibodies 51 Because of the type variability of the e n v gene in the different HIV-1 isolates, most commercial anti-gpl20 mAbs failed to detect this protein by immunoprecipitation. The problem was overcome by using a specific anti-SF2 gpl20 antibody, which was able to immunoprecipitate both gpl20 and its gpl60 precursor (data not shown). The results presented here also revealed a lower antigen expression in most cells infected by the IS - recombinants. The reduced expression was always confirmed by immunofluorescence and fusion assays. Moreover, the numbers of fused HuT 78 lymphocytes observed in the syncytia formation assay were always lower in cells infected by the IS- poxviruses. These data could suggest lower expression of the foreign antigen by either the CP or the FP recombinants lacking the immunosuppressive region, but they may also support the hypothesis of a lower binding capacity for the antibody and for the CD4 receptor of the antigen induced by the IS- poxviruses. In fact, the deletion that we produced in the amino terminus of the gp41 might have induced a higher shedding of the gpl20 from the surface of CPIS-and FPIS--infected cells since the deleted IS region partially overlaps a putative contact region to gpl2052. Furthermore, it has recently been demonstrated that a mutation produced in the N-terminus of gp41 leads to

an attenuated virus that is less cytopathic than the wild-type counterpart both for syncytia formation and for cell lysis 53. The mutation produced in our recombinant is very close to the above-mentioned one, and a similar mechanism may be responsible for the decreased cytopathogenicity observed in in vitro and in vivo infections. Many problems arise in live vaccine preparation and testing for their use in immunocompromised individuals, due to the presence of high levels of neutralizing but non-protective antibodies in the serum of HIV-l-positive patients. The successful development of vaccinia virus as an expression vector 54 and the efficacy of most recombinants in stimulating both humoral and cellmediated immunity increased the interest in CP and FP viruses. In fact, CP and FP viruses are incapable of triggering a productive infection in human cells4s'55, which could be a serious risk in seropositive hosts. Rabbits have also proved to be a suitable animal model for HIV-1 infection56'5s since viral replication occurs, as in chimpanzees, so that a subsequent challenge can be performed. As determined by ELISA, rabbits injected with CP and FP recombinants exhibited a similar immune response, since seroconversion was observed in all injected animals after priming, with high titres soon after the second booster. However, since such a humoral response does not necessarily imply good protection, assays are being performed to detect the presence of neutralizing antibodies. Moreover, the failure of passive immunization to protect chimpanzees from HIV-1 seems to be related, according to Robinson et al. 59, to an IgG serum fraction that is capable, even at very high dilutions, of enhancing the activity of HIV-1 infection. Since the domain that we deleted lies in a region that has already been shown to elicit the production of antibodies enhancing complement-dependent HIV infection (CADE) in vitro 14, it will be interesting to verify whether IS- recombinants show a different efficacy to IS + ones in vivo. ACKNOWLEDGEMENTS The authors are grateful to Dr E. Paoletti (Virogenetics Corporation, Troy, NY) for supplying CP and FP vectors and for permitting one of them (A.R.) to perform the construction of the recombinants using his laboratory facilities. They also gratefully acknowledge Drs J. Tartaglia and C. De Taisne for helpful advice, H. Martinez for synthetic oligonucleotides and Dr M. Gimelli for her contribution to this study. They thank Professor H. Hanafusa (The Rockefeller University, New York), Drs Cattanes and Achilli for helpful suggestions, and NIAID, AIDS Research and Reference Reagent Program for providing HIV-lsv 2 anti-gpl20 antibodies and E-line cells. This research was supported in part by C.N.R., contracts nos. 89.03446.14 and 90.01293.14, and by the Italian Ministry of Health - AIDS Research Project, grant no. 5207.006. A.R. was the recipient of a travel fellowship from the Fulbright CIES. REFERENCES Willey, R.L., Bonifacino, J.S., Potts, B.J., Martin, M.A. and Klausner, R.D. Biosynthesis, cleavage and degradation' of the human immunodeficiency virus 1 envelope glycoprotein gp160. Proc. Natl Acad. Sci. USA 1988, 85, 9580-9584

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