Fusion-Competent Vaccines: Broad Neutralization of Primary Isolates of HIV

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The molecularly cloned envelope gene of ACH168.10 was isolated by polymerase chain reaction with the pCR3.1-Uni plasmid (Invitrogen) [(2, 15);

Tersmette M., et al., J. Virol. 63, 2118 (1989)].

The plasmid encoding expression of the functional 168P envelope protein (168P23) was transfected into COS-7 cells (American Type Culture Collection, Manassas, VA) by calcium phosphate precipitation (20 μg of DNA per 10⁶ cells per 10-cm culture dish) [

Jordan M., Schallhorn A., Wurm F. M., Nucleic Acids Res. 24, 596 (1996);

]. Transiently expressing COS-7 cells were harvested 2 days later with 0.5 mM EDTA in phosphate-buffered saline (PBS), and U87-CD4-CCR5 fusion partners [

Hill C. M., et al., J. Virol. 71, 6296 (1997);

] were prepared with 0.1 mM EDTA in PBS. Cocultures were initiated by mixing the two cell types (1.5 × 10⁶ cells each) in 10-cm culture dishes. The time course of cell-cell fusion was monitored microscopically and by immunochemical staining (HIVIG) in parallel cocultures (15). Cocultures were harvested by formaldehyde fixation at 4 to 5 hours, when little or no overt syncytium formation was evident.

Cultures were fixed in situ with 0.2% formaldehyde in PBS at 4°C overnight [

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]. Cells were subsequently scraped, washed twice with PBS, resuspended at a nominal density of 3 × 10⁶ cells/0.1 ml in PBS containing 10% dimethyl sulfoxide, and frozen at −80°C.

J. D. Scarborough, W. Ellmeier, D. R. Littman, unpublished results. Construction of a CD4 targeted deletion and hu CD4 transgenic mouse has been described [

Killeen N., Sawada S., Littman D. R., EMBO J. 12, 1547 (1993);

]. Briefly, a hu CCR5 transgenic mouse was constructed by molecularly cloning a 1.15-kb hu CCR5 cDNA into an engineered Sal I site in exon 2 of a murine CD4 expression cassette [construct c in

Sawada S., Scarborough J. D., Killeen N., Littman D. R., Cell 77, 917 (1994);

]. This minigene contains the murine CD4 enhancer, the CD4 promoter, the first (noncoding) exon, and intron 1 with an internal deletion that eliminates the CD4 silencer. Transgenic founders were identified by flow cytometry with a monoclonal antibody (mAb) to CCR5. These animals were bred to hu CD4 transgenic mice to yield progeny expressing hu CD4, hu CCR5, and mouse CD4. Pups were screened for expression of hu CD4, hu CCR5, and mouse CD4 by flow cytometry with a Coulter EPICS ELITE flow cytometer. The following antibody reagents were used: mouse antibody to human CD4 conjugated to CyChrome (Pharmingen), mouse antibody to human CCR5 mAb 180 (R&D Systems) with goat antibody to mouse immunoglobulin conjugated to fluorescein isothiocyanate (Caltag), and rat antibody to mouse CD4 L3T4 conjugated to phycoerythrin.

Vaccines comprised formaldehyde-fixed whole cells (3 × 10⁶ cells/0.1 ml) formulated with an equal volume of Ribi adjuvant (R-700; reconstituted in half the recommended volume of PBS); in some experiments, the initial immunization was with adjuvant containing cell wall material (R-730). Mice received 0.05 ml of vaccine in four subcutaneous sites. Booster immunizations were at 3-week intervals, and mice were bled from the tail at 10 to 28 days after immunizations. Ultimately, mice were boosted and exsanguinated by cardiac puncture in order to obtain larger quantities of serum. Animal care was in accordance with institutional guidelines. Serum antibodies directed to gp120 were quantitated by gp120 enzyme-linked immunosorbent assay (ELISA) [

Moore J., Wallace L., Follett E., McKeating J., AIDS 3, 155 (1989)].

This rapid PI virus neutralization assay has been validated relative to our standard neutralization assay in peripheral blood lymphocyte (PBL) culture (15, 17) and performs well in the presence of mouse serum. All sera were heat inactivated before use in neutralization assays.

R. A. LaCasse et al., data not shown. Serum was adsorbed sequentially to protein A Sepharose (Sigma) and protein G agarose (Sigma) at 4°C. Adsorption of antibody was confirmed by gp120 ELISA. The solid supports were combined and antibodies were eluted with 100 mM glycine, pH 2.5. The eluate was neutralized and dialyzed by centrifugal ultrafiltration (Microcon-100; Amicon).

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Envelope-expressing cultures were incubated with sCD4 [

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] (5 μg/ml; 1 hour at 37°C) and subsequently washed to remove unbound sCD4. All FI immunogens were fixed with formaldehyde as described in (10).

Envelope-defective HIV NL4-3-Luc-R⁻E⁻ provirus was pseudotyped with amphotropic MLV envelope protein [

Deng H., et al., Nature 381, 661 (1996)].

A primary isolate of SIVmac251 [

Langlois A. L., et al., J. Virol. 72, 6950 (1998);

] was produced in rhesus PBLs.

A statistical comparison was performed on data comprising all experimental animals and all virus neutralization assays. A simple model for virus-antibody binding was used to calculate a “binding constant” K for each assay, and a mean K value was determined for each mouse. Two-sample unpooled t tests were performed on log-transformed K values to compare groups pairwise. Bonferroni-adjusted comparison demonstrated a significant difference in mean neutralization between FC and FI immunogens (P = 0.001). This analysis included 26 independent neutralization assays of FC sera, from all mice receiving FC immunogen, and similarly, 35 assays of FI sera. In all experiments, responses within experimental groups were consistent and uniform.

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], SHIV89.6, and SHIV89.6P [

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] were provided with permission by D. Montefiori (Duke University Medical Center). All other primary isolates were obtained through the NIH AIDS Research and Reference Reagent Program and the UNAIDS Network for HIV-1 Isolation and Characterization. PI viruses were subjected to limited expansion in phytohemagglutinin-activated PBLs (2).

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FC vaccine serum (1:10 dilution in cell culture medium) was sequentially adsorbed four times with ∼10⁶ formaldehyde-fixed COS cells expressing 168P envelope protein. Incubations were for 1 hour at 4°C with rocking. Controls included prebleed serum and formaldehyde-fixed, mock-transfected COS cells. Adsorption of bulk anti-gp120 was monitored by gp120 ELISA. Final sera were tested for neutralization of HIV 168P with U87-CD4-CXCR4 cells. Parallel studies with intact but nonfixed cells yielded concordant results.

Pooled FC and FI sera (1:8 dilution) were incubated with attached U87-CD4-CCR5 cells (2 × 10⁴ cells per 96-well microculture) for 1 hour at 37°C with occasional mixing. Supernatants were tested for neutralization of HIV 168P with U87-CD4-CCR5 cells.

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J.H.N. was supported by targeted research grant 02560-23-RGV from the American Foundation for AIDS Research (AmFAR), funded in part by Concerned Parents for AIDS Research (CPFA). Additional funds were provided by The University of Montana, NIH AREA grant AI41165, and the M. J. Murdock Charitable Trust. D.R.L. was supported by NIH grant AI33856 and by a grant from AmFAR and is an investigator of the Howard Hughes Medical Institute. We thank E. Walker (Ribi ImmunoChem Research, Incorporated, Hamilton, MT) and L. Griggs for flow cytometry, C. Mackay (Leukosite) and M. Tsang for CCR5 mAbs, W. Ellmeier for the transgenic mouse CCR5 construct, D. Montefiori (Duke University Medical Center) for providing SIVmac251, HIV 89.6, and related SHIV viruses, and C. Weiss (U.S. Food and Drug Administration) for providing amphotropic MLV envelope protein pseudotyped HIV virions. Primary HIV isolates and other reagents were obtained from the NIH AIDS Research and Reference Reagent Program, the NIBSC (UK) AIDS Reagent Program, and the UNAIDS Network for HIV-1 Isolation and Characterization. We are grateful to D. A. Patterson (University of Montana, Department of Mathematics) for statistical analysis of neutralization data, and to J. Moore and D. Montefiori for constructive review of the manuscript. Discussions with C. Barbas III and E. Berger were important in the development of these studies.