Journal of Immunological Methods, 140 (1991) 135-138
135
© 1991 Elsevier Science Publishers B.V. 0022-1759/91/$03.50 ADONIS 002217599100203U
JIM 05985
Letter to the editors
Generation of antibodies against recombinant HIV-gpl20 antigen through a novel immunization procedure D e b o r a h L. Matour, R o b e r t K. Clark and Z d e n k a L. J o n a k Department of Cell Sciences, SmithKline Beecham Pharmaceuticals, 709 Swedeland Road, King of Prussia, PA 19406-2799, U.S.A. (Received 11 December 1990, revised received 22 March 1991, accepted 22 March 1991)
Dear Editors, This report describes a method for generating polyclonal antibodies against HIV-gpl20 by employing transfected Chinese hamster ovary (CHO) cells, expressing recombinant gpl20, as a source of immunogen. Human immunodeficiency virus (HIV), the etiologic agent of AIDS, expresses envelope molecules gpl20 (extracellular) and gp41 (transmembrane) that serve as potential targets for intervention against viral infection. The selectivity of the virus for CD4-bearing cells results from interactions between gpl20 and a high affinity binding site of CD4 (McDougal et al., 1986); the CD4 molecule acts as a receptor for the virus (Dagleish et al., 1984). In addition, the formation of syncytia by fusion of infected cells with CD4positive cells is mediated, at least in part, by the envelope proteins expressed on the surface of infected cells (Sodroski et al., 1986). The immunogenicity of the HIV-1 envelope is evident since antibodies (Abs) against gpl20 are generated in infected individuals (Weiss et al., 1985). Immune responses against gpl20 can also be experimentally induced in animals (Robey et al., 1986; Nara et al., 1987; Ho et al., 1988). The ability of gpl20 to induce an immune response may be exploited by developing antibodies against the envelope proteins. Such reagents can be use-
Correspondence to: Z.L. Jonak, SmithKline Beecham Pharmaceuticals, Department of Cell Sciences (L109), 709 Swedeland Road, King of Prussia, PA 19406, U.S.A.
ful as vaccine candidates and as therapeutic or prophylactic agents that block syncytia formation or viral penetration. Antibodies could also be useful diagnostic reagents and research tools in studying gpl20-CD4 interactions. The ability of the immunogen to elicit an immune response is essential for Ab production. Several studies showed that Abs can be generated against viral lysates (Broliden et al., 1990), virally infected ceils (Fung et al., 1987), peptides (Dagleish et al., 1988; Ho et al., 1988), and recombinant proteins (Veronese et al., 1989). When recombinant proteins are used as immunogens for antibody (Ab) generation, several steps have to be optimized prior to immunization. First, suitable expression levels of recombinant proteins must be achieved (the expression system must be modified). It is often difficult to obtain recombinant protein in soluble form. In many cases, insolubility of recombinant proteins can result in delays in obtaining sufficient quantities of antigen in a form acceptable for use as an immunogen. Second, the antigen requires purification to homogeneity by standard techniques (e.g., column chromatography, differential extraction, subcellular fractionation a n d / o r electrophoresis). Such manipulations present several obstacles. Purification procedures may be very complex, resulting in a low degree of purity and only small quantities of recoverable purified antigen. Changes in the native conformation of the antigen molecules can result in the loss of an important epitope or the generation of 'new' altered epitopes that are not present on the native antigen. We eliminated the
136 2°0
'
Mouse 1 •
Mouse 2
Mouse 3
1o5 "
Mouse 4 Mouse 5 Mouse 6 Background
1.0"
o
0.5
>,
•"=-
0.0
p, Q
tJ
Rabbit 1
Rabbit 2
2.0
Background
Q.
o
1.5"
1°0 "
0.5
0.0 100
. . . . . . . .
,
. . . . . . . .
1000
Dilution
i
10000
Factor
. . . . . . .
10
000
(log)
Fig. 1. Final serum titers from immunized animals were screened by anti-gpl20 ELISA (described in the text). The sera were serially diluted and added to the plates. The data are plotted as serial dilutions vs. absorbance at 450 nm. Each line represents the results from an individual animal. The background is an average of the titers from pre-immune serum. A: mice 1-3 were immunized a total of six times; mice 4-6 were immunized a total of eight times. B: both rabbits were immunized six times. time-consuming tasks of optimizing the expression systems and modifying steps in purification by directly using viable cells as an immunogen. Our results suggest that direct immunization with transfected cells expedites the production of antibodies against recombinant proteins. Rabbits (New Zealand White females) and mice ( B A L B / c females) were immunized with viable recombinant C H O cells (TF182) which were expressing low levels of HIV-gpl20. This cell line was generated by transfecting C H O cells with plasmid containing R O U S - L T R - g p l 2 0 /3globin-DHFR genes. The transfection was performed by electroporation, and the resulting clone was selected and amplified for gpl20 expression
by methotrexate (80 nM). Confluent cultures secrete 30 n g / m l of HIV-gpl20. The cells were treated with trypsin, washed and resuspended in medium (F12 medium supplemented with 10% fetal bovine serum, 2 nM glutamine, 10 /xg/ml gentamycin). The rabbits were inoculated subcutaneously in multiple sites with a total of 5 x 107 cells. Immunization was performed approximately every 2 months for a total of six immunizations. Serum titers were monitored using the anti-gpl20 ELISA. Final immunization was performed with shredded viable ceils instead of whole cells. The mice were inoculated intraperitoneally with 7 x 10 6 cells and subcutaneously with 3 x 10 6 cells. Inoculations were repeated approximately every 2
137 weeks. A total of four to six immunizations were performed prior to screening the serum for antigpl20 antibody specificity in the ELISA assay. Purified recombinant gpl20 protein, expressed in Drosophila cells, was used as an antigen for the detection of anti-gpl20 activity in the ELISA assay. We speculated that some epitopes expressed on CHO-gpl20 will be common to Drosophila-gpl20 and therefore polyclonal antibody responses against TF182 cells can be evaluated by using the purified Drosopohila recombinant protein. The antigen was absorbed overnight at 4°C onto Immulon-II microplate wells (Dynatech) at a concentration of 100 ng/well in 100 mM NaHCO 3, pH 9.6. All the washes were done with PBS Tween 20 (phosphate-buffered saline, 0.05% Tween 20). The wells were washed once and blocked for 1 h with 1% bovine serum albumin (Sigma) in PBS with 0.05% sodium azide. After additional wash, serum samples were added to the wells, incubated for 2 h at room temperature, and unbound protein was removed by repeated washing (four times). Antibodies were detected with peroxidase-labeled goat anti-mouse IgG, IgM or goat anti-rabbit IgG, IgM (Boehringer-Mannheim) diluted 1/2000 or 1/5000, respectively, in PBS-Tween 20. The wells were washed four times and developed by 10 min incubation with 10 mg/ml substrate o-phenylenediamine (Kodak) in 100 nM citrate buffer, pH 4.5 and 12% H 2 0 2. After the reaction was stopped with 100 nM sodium fluoride, the absorbance was determined at 460 nm on a Dynatech ELISA reader. The murine polyclonal antibody response against HIV-gpl20 from TF182 cells is presented in Fig. 1A. All six mice developed a specific immune response against the immunogen. Interestingly, mice 4-6, which were immunized a total of eight times, appeared to have a higher response than mice 1-3, which were immunized six times. The amount of serum obtained from these mice was very limited, and therefore only specificity for anti-gpl20 antibodies was tested in the ELISA assay. In addition, spleens from immunized animals were used to generate hybridomas by fusing them with SP2/0 myeloma cells, using the modified method of Kennett (Kennett et al., 1980). From two fusions we obtained a total of 1237 clones, out of which 83 (6.7%) produced
monoclonal antibodies with anti-gp120 binding specificity (data not shown). Rabbit polyclonal response against viable TF182 cells is shown in Fig. lB. Both rabbits developed high antibody titers against gpl20. The non-specific control protein (recombinant tissue plasminogen activator) showed no rabbit polyclonal antibody binding. The specificity of the antibodies against gpl20 were further supported by: (1) Western blot analysis in which immunoreactivity was detected against HIV-viral lysates with a specific band at 120 kDa; (2) by inhibition of syncytia formation indicating the potential of these antibodies to neutralize the fusion process between infected cells (gpl20) and CD4 + cells; (3) by immunocytochemistry, demonstrating that the antibodies selectively stain TF182 cells as compared to CHO controls. To further examine the ability of rabbit polyclonal antibodies to recognize gpl20, we added gpl20 (purified from TF182 cells) to CD4 positive cells and allowed binding of gpl20 to the CD4 receptor. Following washing, antibodies bound to gpl20 were detected with fluorescently labeled goat anti-rabbit Ig. The results showed cell surface staining with specific antisera, and the preimmune serum showed no binding to gpl20 in this system (data not shown). The data presented in Fig. 1 indicate that polyclonal antibodies can be generated to HIVgpl20 protein by immunizing animals with a viable CHO recombinant cell line expressing/ secreting low levels of gpl20 protein. This method of immunization alleviates the need for amplification (increase in the level of protein expression), isolation, and purification of protein as well as the need to find an adjuvant suitable for stimulation of the immune system. In addition, the immunogen is presented to the animal's immune system in unaltered biological configuration, resuiting in antibodies that detect gpl20 protein only in its native form (i.e., fixed or denatured gpl20 have limited immunoreactivity). This minimizes the chance of destroying important epitopes or creating irrelevant ones during the manipulation process. One can speculate that after immunization the cells will secrete gpl20 for a short time before the host immune system will destroy the cells, at which time cytoplasmic gpl20
138 serves as a f u r t h e r i m m u n o s t i m u l a n t . This m e t h o d of i m m u n i z a t i o n may be a p p l i e d to o t h e r recomb i n a n t p r o t e i n s expressed by cells. A l t h o u g h T F 1 8 2 cells express g p l 2 0 intracellularly prior to secretion, t r a n s f e c t e d cells expressing a p r o t e i n o n the cell surface should also provide a good i m m u n o g e n . A s long as the cells are foreign to the host, t h e r e should be n o n e e d for a traditional a d j u v a n t in i n d u c i n g a n i m m u n e response. However, a d d i n g a n a d j u v a n t may boost the response in some systems. O t h e r a d a p t a t i o n s of the p r o c e d u r e may also e n h a n c e a n t i b o d y p r o d u c tion. F o r example, i m m u n i z i n g with i r r a d i a t e d or sonicated, a n d / o r i m m u n i z i n g m o r e times may i n d u c e a p a n e l of a n t i b o d i e s with varied specificities. I n s u m m a r y , o u r results i n d i c a t e that the prod u c t i o n of a n t i b o d i e s against H I V - g p l 2 0 can be facilitated by direct i m m u n i z a t i o n with viable rec o m b i n a n t cells expressing H I V - g p l 2 0 .
Acknowledgements W e t h a n k H. J o h a n s e n for the plasmid, J. Culp for purified D r o s o p h i l a g p l 2 0 , A. C o n s t a n t i n e s c u for testing the a n t i b o d i e s o n W e s t e r n blot, a n d S. Trulli a n d E. H e n r i for testing the a n t i b o d i e s in the syncytia assay. W e also t h a n k R. G r e i g for providing us with v a l u a b l e criticism a n d for his s u p p o r t o n this project.
References Broliden, P.A., Ljunggren, K., Hinkula, J., Norrby, E., Akerblom, L. and Wahren, B. (1990) A monoclonal antibody to human immunodeficiency virus type 1 which mediates cellular cytotoxicity and neutralization. J. Virol. 64, 936-940. Dagleish, A.G., Beverly, P.C., Clapham, P.R., Crawford, D.H., Greaves, M.F. and Weiss, R.A. (1984) The CD4 (T4) antigen is an essential component of the receptor for the AIDS retrovirus. Nature 312, 763-766.
Dagleish, A.G., Chanh, T.C., Kennedy, R.C., Kanda, P., Clapham, P.R. and Weiss, R.A. (1988) Neutralization of diverse HIV-1 strains by monoclonal antibodies raised against a gp41 synthetic peptide. Virology 165, 209-215. Fung, M.S.C., Sun, C., Sun, N., Chang, N.T. and Chang, T.W. (1987) Monoclonal antibodies that neutralize HIV-1 virions and inhibit syncytium formation by infected cells. Biotechnology 5, 940-946. Ho, D.A., Kaplan, J.C., Rackausjas, I.E. and Gurney, M.E. (1988) Second conserved domain of gpl20 is important for HIV infectivity and antibody neutralization. Science 239, 1021-1023. Kennett, R.H., McKearn, T.J. and Bechtol, K.B. (Eds.) (1980) In: Monoclonal Antibodies. Hybridomas: A New Dimension in Biological Analyses. Plenum Press, New York, pp. 365-367. McDougal, J.S., Nicholson, K.A., Cross, G.D., Cort, S.P., Kennedy, M.S. and Mawle, A.C. (1986) Binding of the human retrovirus HTLV-III/LAV/ARV/HIV to the CD4 (T4) molecule: conformation dependence, epitope mapping, antibody inhibition, and potential for idiotypic mimicry. J. Immunol. 137, 2937-2944. Nara, P.L., Robey, W.G., Gonda, M.A., Carter, S.G. and Fischinger, P.J. (1987) Absence of cytotoxic antibody to human immunodeficiency virus infected cells in humans and its induction in animals after infection or immunization with purified envelope glycoprotein gpl20. Proc. Natl. Acad. Sci. U.S.A. 84, 3797-3801. Robey, W.D,, Arthur, L.O., Manhews, T.J., Langois, A., Copeland, T.D., Lerche, N.W., Orsozlan, S., Bolognesi, D.P., Gilden, R.V. and Fischinger, P.J. (1986) Prospect for prevention of human immunodeficiency virus infection: purified 120 kDa envelope glycoprotein induces neutralizing antibody. Proc. Natl. Acad. Sci. U.S.A. 83, 7023-7027. Sodroski, J., Goh, W.C., Rosen, C., Campbell, K. and Haselfine, W.A. (1986) Role of the HTLV-III/LAV envelope in syncytium formation and cytopathicity. Nature 322, 470474. Veronese, F.D., Rahman, R., Kalyanaraman, V.S., Pal, R., Lusso, P., Tritch, R., Petteway, S., Gallo, R.C. and Sarngadharan, M.D. (1989) Monoclonal antibodies to HTLVllla51 gp41: Delineation of an immunoreactive conserved epitope in the transmembrane region of divergent isolates of HIV-1. AIDS Res. H. Retro. 5, 479-486. Weiss, R.A., Clapham, P.R., Cheingsong-Popov, R., Dagleish, A.G., Carrie, C.A., Weller, I.V.D. and Tedder, R.S. (1985) Neutralization of human T-lymphotrophic virus type III by sera of AIDS and AIDS-risk patients. Nature 316, 69-72.
135
© 1991 Elsevier Science Publishers B.V. 0022-1759/91/$03.50 ADONIS 002217599100203U
JIM 05985
Letter to the editors
Generation of antibodies against recombinant HIV-gpl20 antigen through a novel immunization procedure D e b o r a h L. Matour, R o b e r t K. Clark and Z d e n k a L. J o n a k Department of Cell Sciences, SmithKline Beecham Pharmaceuticals, 709 Swedeland Road, King of Prussia, PA 19406-2799, U.S.A. (Received 11 December 1990, revised received 22 March 1991, accepted 22 March 1991)
Dear Editors, This report describes a method for generating polyclonal antibodies against HIV-gpl20 by employing transfected Chinese hamster ovary (CHO) cells, expressing recombinant gpl20, as a source of immunogen. Human immunodeficiency virus (HIV), the etiologic agent of AIDS, expresses envelope molecules gpl20 (extracellular) and gp41 (transmembrane) that serve as potential targets for intervention against viral infection. The selectivity of the virus for CD4-bearing cells results from interactions between gpl20 and a high affinity binding site of CD4 (McDougal et al., 1986); the CD4 molecule acts as a receptor for the virus (Dagleish et al., 1984). In addition, the formation of syncytia by fusion of infected cells with CD4positive cells is mediated, at least in part, by the envelope proteins expressed on the surface of infected cells (Sodroski et al., 1986). The immunogenicity of the HIV-1 envelope is evident since antibodies (Abs) against gpl20 are generated in infected individuals (Weiss et al., 1985). Immune responses against gpl20 can also be experimentally induced in animals (Robey et al., 1986; Nara et al., 1987; Ho et al., 1988). The ability of gpl20 to induce an immune response may be exploited by developing antibodies against the envelope proteins. Such reagents can be use-
Correspondence to: Z.L. Jonak, SmithKline Beecham Pharmaceuticals, Department of Cell Sciences (L109), 709 Swedeland Road, King of Prussia, PA 19406, U.S.A.
ful as vaccine candidates and as therapeutic or prophylactic agents that block syncytia formation or viral penetration. Antibodies could also be useful diagnostic reagents and research tools in studying gpl20-CD4 interactions. The ability of the immunogen to elicit an immune response is essential for Ab production. Several studies showed that Abs can be generated against viral lysates (Broliden et al., 1990), virally infected ceils (Fung et al., 1987), peptides (Dagleish et al., 1988; Ho et al., 1988), and recombinant proteins (Veronese et al., 1989). When recombinant proteins are used as immunogens for antibody (Ab) generation, several steps have to be optimized prior to immunization. First, suitable expression levels of recombinant proteins must be achieved (the expression system must be modified). It is often difficult to obtain recombinant protein in soluble form. In many cases, insolubility of recombinant proteins can result in delays in obtaining sufficient quantities of antigen in a form acceptable for use as an immunogen. Second, the antigen requires purification to homogeneity by standard techniques (e.g., column chromatography, differential extraction, subcellular fractionation a n d / o r electrophoresis). Such manipulations present several obstacles. Purification procedures may be very complex, resulting in a low degree of purity and only small quantities of recoverable purified antigen. Changes in the native conformation of the antigen molecules can result in the loss of an important epitope or the generation of 'new' altered epitopes that are not present on the native antigen. We eliminated the
136 2°0
'
Mouse 1 •
Mouse 2
Mouse 3
1o5 "
Mouse 4 Mouse 5 Mouse 6 Background
1.0"
o
0.5
>,
•"=-
0.0
p, Q
tJ
Rabbit 1
Rabbit 2
2.0
Background
Q.
o
1.5"
1°0 "
0.5
0.0 100
. . . . . . . .
,
. . . . . . . .
1000
Dilution
i
10000
Factor
. . . . . . .
10
000
(log)
Fig. 1. Final serum titers from immunized animals were screened by anti-gpl20 ELISA (described in the text). The sera were serially diluted and added to the plates. The data are plotted as serial dilutions vs. absorbance at 450 nm. Each line represents the results from an individual animal. The background is an average of the titers from pre-immune serum. A: mice 1-3 were immunized a total of six times; mice 4-6 were immunized a total of eight times. B: both rabbits were immunized six times. time-consuming tasks of optimizing the expression systems and modifying steps in purification by directly using viable cells as an immunogen. Our results suggest that direct immunization with transfected cells expedites the production of antibodies against recombinant proteins. Rabbits (New Zealand White females) and mice ( B A L B / c females) were immunized with viable recombinant C H O cells (TF182) which were expressing low levels of HIV-gpl20. This cell line was generated by transfecting C H O cells with plasmid containing R O U S - L T R - g p l 2 0 /3globin-DHFR genes. The transfection was performed by electroporation, and the resulting clone was selected and amplified for gpl20 expression
by methotrexate (80 nM). Confluent cultures secrete 30 n g / m l of HIV-gpl20. The cells were treated with trypsin, washed and resuspended in medium (F12 medium supplemented with 10% fetal bovine serum, 2 nM glutamine, 10 /xg/ml gentamycin). The rabbits were inoculated subcutaneously in multiple sites with a total of 5 x 107 cells. Immunization was performed approximately every 2 months for a total of six immunizations. Serum titers were monitored using the anti-gpl20 ELISA. Final immunization was performed with shredded viable ceils instead of whole cells. The mice were inoculated intraperitoneally with 7 x 10 6 cells and subcutaneously with 3 x 10 6 cells. Inoculations were repeated approximately every 2
137 weeks. A total of four to six immunizations were performed prior to screening the serum for antigpl20 antibody specificity in the ELISA assay. Purified recombinant gpl20 protein, expressed in Drosophila cells, was used as an antigen for the detection of anti-gpl20 activity in the ELISA assay. We speculated that some epitopes expressed on CHO-gpl20 will be common to Drosophila-gpl20 and therefore polyclonal antibody responses against TF182 cells can be evaluated by using the purified Drosopohila recombinant protein. The antigen was absorbed overnight at 4°C onto Immulon-II microplate wells (Dynatech) at a concentration of 100 ng/well in 100 mM NaHCO 3, pH 9.6. All the washes were done with PBS Tween 20 (phosphate-buffered saline, 0.05% Tween 20). The wells were washed once and blocked for 1 h with 1% bovine serum albumin (Sigma) in PBS with 0.05% sodium azide. After additional wash, serum samples were added to the wells, incubated for 2 h at room temperature, and unbound protein was removed by repeated washing (four times). Antibodies were detected with peroxidase-labeled goat anti-mouse IgG, IgM or goat anti-rabbit IgG, IgM (Boehringer-Mannheim) diluted 1/2000 or 1/5000, respectively, in PBS-Tween 20. The wells were washed four times and developed by 10 min incubation with 10 mg/ml substrate o-phenylenediamine (Kodak) in 100 nM citrate buffer, pH 4.5 and 12% H 2 0 2. After the reaction was stopped with 100 nM sodium fluoride, the absorbance was determined at 460 nm on a Dynatech ELISA reader. The murine polyclonal antibody response against HIV-gpl20 from TF182 cells is presented in Fig. 1A. All six mice developed a specific immune response against the immunogen. Interestingly, mice 4-6, which were immunized a total of eight times, appeared to have a higher response than mice 1-3, which were immunized six times. The amount of serum obtained from these mice was very limited, and therefore only specificity for anti-gpl20 antibodies was tested in the ELISA assay. In addition, spleens from immunized animals were used to generate hybridomas by fusing them with SP2/0 myeloma cells, using the modified method of Kennett (Kennett et al., 1980). From two fusions we obtained a total of 1237 clones, out of which 83 (6.7%) produced
monoclonal antibodies with anti-gp120 binding specificity (data not shown). Rabbit polyclonal response against viable TF182 cells is shown in Fig. lB. Both rabbits developed high antibody titers against gpl20. The non-specific control protein (recombinant tissue plasminogen activator) showed no rabbit polyclonal antibody binding. The specificity of the antibodies against gpl20 were further supported by: (1) Western blot analysis in which immunoreactivity was detected against HIV-viral lysates with a specific band at 120 kDa; (2) by inhibition of syncytia formation indicating the potential of these antibodies to neutralize the fusion process between infected cells (gpl20) and CD4 + cells; (3) by immunocytochemistry, demonstrating that the antibodies selectively stain TF182 cells as compared to CHO controls. To further examine the ability of rabbit polyclonal antibodies to recognize gpl20, we added gpl20 (purified from TF182 cells) to CD4 positive cells and allowed binding of gpl20 to the CD4 receptor. Following washing, antibodies bound to gpl20 were detected with fluorescently labeled goat anti-rabbit Ig. The results showed cell surface staining with specific antisera, and the preimmune serum showed no binding to gpl20 in this system (data not shown). The data presented in Fig. 1 indicate that polyclonal antibodies can be generated to HIVgpl20 protein by immunizing animals with a viable CHO recombinant cell line expressing/ secreting low levels of gpl20 protein. This method of immunization alleviates the need for amplification (increase in the level of protein expression), isolation, and purification of protein as well as the need to find an adjuvant suitable for stimulation of the immune system. In addition, the immunogen is presented to the animal's immune system in unaltered biological configuration, resuiting in antibodies that detect gpl20 protein only in its native form (i.e., fixed or denatured gpl20 have limited immunoreactivity). This minimizes the chance of destroying important epitopes or creating irrelevant ones during the manipulation process. One can speculate that after immunization the cells will secrete gpl20 for a short time before the host immune system will destroy the cells, at which time cytoplasmic gpl20
138 serves as a f u r t h e r i m m u n o s t i m u l a n t . This m e t h o d of i m m u n i z a t i o n may be a p p l i e d to o t h e r recomb i n a n t p r o t e i n s expressed by cells. A l t h o u g h T F 1 8 2 cells express g p l 2 0 intracellularly prior to secretion, t r a n s f e c t e d cells expressing a p r o t e i n o n the cell surface should also provide a good i m m u n o g e n . A s long as the cells are foreign to the host, t h e r e should be n o n e e d for a traditional a d j u v a n t in i n d u c i n g a n i m m u n e response. However, a d d i n g a n a d j u v a n t may boost the response in some systems. O t h e r a d a p t a t i o n s of the p r o c e d u r e may also e n h a n c e a n t i b o d y p r o d u c tion. F o r example, i m m u n i z i n g with i r r a d i a t e d or sonicated, a n d / o r i m m u n i z i n g m o r e times may i n d u c e a p a n e l of a n t i b o d i e s with varied specificities. I n s u m m a r y , o u r results i n d i c a t e that the prod u c t i o n of a n t i b o d i e s against H I V - g p l 2 0 can be facilitated by direct i m m u n i z a t i o n with viable rec o m b i n a n t cells expressing H I V - g p l 2 0 .
Acknowledgements W e t h a n k H. J o h a n s e n for the plasmid, J. Culp for purified D r o s o p h i l a g p l 2 0 , A. C o n s t a n t i n e s c u for testing the a n t i b o d i e s o n W e s t e r n blot, a n d S. Trulli a n d E. H e n r i for testing the a n t i b o d i e s in the syncytia assay. W e also t h a n k R. G r e i g for providing us with v a l u a b l e criticism a n d for his s u p p o r t o n this project.
References Broliden, P.A., Ljunggren, K., Hinkula, J., Norrby, E., Akerblom, L. and Wahren, B. (1990) A monoclonal antibody to human immunodeficiency virus type 1 which mediates cellular cytotoxicity and neutralization. J. Virol. 64, 936-940. Dagleish, A.G., Beverly, P.C., Clapham, P.R., Crawford, D.H., Greaves, M.F. and Weiss, R.A. (1984) The CD4 (T4) antigen is an essential component of the receptor for the AIDS retrovirus. Nature 312, 763-766.
Dagleish, A.G., Chanh, T.C., Kennedy, R.C., Kanda, P., Clapham, P.R. and Weiss, R.A. (1988) Neutralization of diverse HIV-1 strains by monoclonal antibodies raised against a gp41 synthetic peptide. Virology 165, 209-215. Fung, M.S.C., Sun, C., Sun, N., Chang, N.T. and Chang, T.W. (1987) Monoclonal antibodies that neutralize HIV-1 virions and inhibit syncytium formation by infected cells. Biotechnology 5, 940-946. Ho, D.A., Kaplan, J.C., Rackausjas, I.E. and Gurney, M.E. (1988) Second conserved domain of gpl20 is important for HIV infectivity and antibody neutralization. Science 239, 1021-1023. Kennett, R.H., McKearn, T.J. and Bechtol, K.B. (Eds.) (1980) In: Monoclonal Antibodies. Hybridomas: A New Dimension in Biological Analyses. Plenum Press, New York, pp. 365-367. McDougal, J.S., Nicholson, K.A., Cross, G.D., Cort, S.P., Kennedy, M.S. and Mawle, A.C. (1986) Binding of the human retrovirus HTLV-III/LAV/ARV/HIV to the CD4 (T4) molecule: conformation dependence, epitope mapping, antibody inhibition, and potential for idiotypic mimicry. J. Immunol. 137, 2937-2944. Nara, P.L., Robey, W.G., Gonda, M.A., Carter, S.G. and Fischinger, P.J. (1987) Absence of cytotoxic antibody to human immunodeficiency virus infected cells in humans and its induction in animals after infection or immunization with purified envelope glycoprotein gpl20. Proc. Natl. Acad. Sci. U.S.A. 84, 3797-3801. Robey, W.D,, Arthur, L.O., Manhews, T.J., Langois, A., Copeland, T.D., Lerche, N.W., Orsozlan, S., Bolognesi, D.P., Gilden, R.V. and Fischinger, P.J. (1986) Prospect for prevention of human immunodeficiency virus infection: purified 120 kDa envelope glycoprotein induces neutralizing antibody. Proc. Natl. Acad. Sci. U.S.A. 83, 7023-7027. Sodroski, J., Goh, W.C., Rosen, C., Campbell, K. and Haselfine, W.A. (1986) Role of the HTLV-III/LAV envelope in syncytium formation and cytopathicity. Nature 322, 470474. Veronese, F.D., Rahman, R., Kalyanaraman, V.S., Pal, R., Lusso, P., Tritch, R., Petteway, S., Gallo, R.C. and Sarngadharan, M.D. (1989) Monoclonal antibodies to HTLVllla51 gp41: Delineation of an immunoreactive conserved epitope in the transmembrane region of divergent isolates of HIV-1. AIDS Res. H. Retro. 5, 479-486. Weiss, R.A., Clapham, P.R., Cheingsong-Popov, R., Dagleish, A.G., Carrie, C.A., Weller, I.V.D. and Tedder, R.S. (1985) Neutralization of human T-lymphotrophic virus type III by sera of AIDS and AIDS-risk patients. Nature 316, 69-72.
