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. 2005 Dec;79(23):14804-14.
doi: 10.1128/JVI.79.23.14804-14814.2005.

Induction of neutralizing antibodies against human immunodeficiency virus type 1 primary isolates by Gag-Env pseudovirion immunization

Jason Hammonds  1 , Xuemin Chen, Timothy Fouts, Anthony DeVico, David Montefiori, Paul Spearman
Affiliations

Induction of neutralizing antibodies against human immunodeficiency virus type 1 primary isolates by Gag-Env pseudovirion immunization

Jason Hammonds et al. J Virol. 2005 Dec.

Abstract

A major challenge for the development of an effective HIV vaccine is to elicit neutralizing antibodies against a broad array of primary isolates. Monomeric gp120-based vaccine approaches have not been successful in inducing this type of response, prompting a number of approaches designed to recreate the native glycoprotein complex that exists on the viral membrane. Gag-Env pseudovirions are noninfectious viruslike particles that recreate the native envelope glycoprotein structure and have the potential to generate neutralizing antibody responses against primary isolates. In this study, an inducible cell line was created in order to generate Gag-Env pseudovirions for examination of neutralizing antibody responses in guinea pigs. Unadjuvanted pseudovirions generated relatively weak anti-gp120 responses, while the use of a block copolymer water-in-oil emulsion or aluminum hydroxide combined with CpG oligodeoxynucleotides resulted in high levels of antibodies that bind to gp120. Sera from immunized animals neutralized a panel of human immunodeficiency virus (HIV) type 1 primary isolate viruses at titers that were significantly higher than that of the corresponding monomeric gp120 protein. Interpretation of these results was complicated by the occurrence of neutralizing antibodies directed against cellular (non-envelope protein) components of the pseudovirion. However, a major component of the pseudovirion-elicited antibody response was directed specifically against the HIV envelope. These results provide support for the role of pseudovirion-based vaccines in generating neutralizing antibodies against primary isolates of HIV and highlight the potential confounding role of antibodies directed at non-envelope cell surface components.

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Figures

FIG. 1.
FIG. 1.
Production of Gag-Env pseudovirions by XC-18 cell line. Cells were induced by addition of 2 μg/ml doxycycline (Dox). (A) p24 antigen release in cellular supernatants was measured at the times indicated. Closed squares indicate induced cell culture, and open triangles indicate uninduced cells. (B) Envelope glycoprotein secretion into cellular supernatants harvested at time points following induction as measured by gp120 capture ELISA. (C) Determination of sedimentation pattern of pseudovirions harvested from induced XC-18 supernatants by sucrose density-equilibrium gradient analysis. Metabolically labeled, doxycycline-induced XC-18 cell supernatants were concentrated through a 20% sucrose cushion, resuspended, and subjected to centrifugation on 20 to 60% sucrose gradients. Fractions were collected from the top of the gradient and immunoprecipitated using pooled HIV-positive patients' sera prior to analysis by SDS-polyacrylamide gel electrophoresis and autoradiography.
FIG. 2.
FIG. 2.
Electron micrographs of immature HIV-1 VLPs produced from doxycycline-induced XC-18 cellular supernatants. Supernatants were concentrated through 20% sucrose cushion. Results from examination of pelleted VLPs are shown. (A) Magnification, ×49,500; bar = 500 nm. (B) Magnification, ×105,000; bar = 100 nm.
FIG. 3.
FIG. 3.
Schematic diagram of guinea pig immunization protocol. Frequency and distribution of inoculations and bleeds are indicated for each of four groups (A to D). Within individual groups, a horizontal line indicates repeated administration of the same immunogen. A vertical bar denotes termination of the group protocol. TMx, TiterMax Gold; ID, intradermal.
FIG. 4.
FIG. 4.
gp120-specific antibodies raised by pseudovirion and soluble envelope glycoprotein inoculations were quantified by endpoint antibody (Ab) binding titration. (A) Anti-gp120 endpoint antibody titers for guinea pigs immunized with 5 μg of particle-associated BaL gp120 adjuvanted with TiterMax Gold (TMx) and for unadjuvanted pseudovirions. (B) Anti-gp120 endpoint antibody titers for guinea pigs immunized with 5 μg of particle-associated BaL gp120 adjuvanted with alum and CpG ODN sequence 2007. (C) Anti-gp120 endpoint titers for guinea pigs immunized with 5 μg of particle-associated BaL gp120 adjuvanted with alum/CpG ODN 2007 for the first two inoculations at weeks 0 and 2 followed by subsequent inoculations with 5 μg of soluble BaL gp120 adjuvanted with TiterMax Gold. (D) Endpoint antibody titers for guinea pigs immunized with 5 μg of soluble BaL gp120 adjuvanted with TiterMax Gold.
FIG. 5.
FIG. 5.
Neutralization of HIV-1 BaL, SS1196, and Pvo by sera generated from guinea pig immunization protocol. Titrations of neutralizing activity were measured using a single-round infectivity assay in TZM-bl cells and quantitated by a reduction in RLU. Fifty-percent neutralization activity levels are indicated by horizontal gray lines. Neutralizing antibody titers are expressed as the serum dilutions required to reduce RLU by 50%. TMx, TiterMax Gold.
FIG. 6.
FIG. 6.
Neutralization of an amphotropic MLV Env-pseudotyped HIV-1 virus by sera generated from guinea pig immunization regimens. Titrations of neutralizing activity were measured using a single-round infectivity assay in TZM-bl cells and quantitated by a reduction in RLU. Fifty-percent neutralization activity levels are indicated by horizontal gray lines. Neutralizing antibody titers are expressed as the serum dilutions required to reduce RLU by 50%. TMx, TiterMax Gold.
FIG. 7.
FIG. 7.
Specificities of neutralization of HIV-1 BaL by sera generated from guinea pig immunization protocol. Titrations of neutralizing activity were measured using a single-round infectivity assay in TZM-bl cells and quantitated by a reduction in RLU. (A) Neutralization activity of prebleed (triangles) and terminal bleed (squares) serum samples from a representative guinea pig from each pseudovirion immunization group. The representatives from each group had the greatest anti-gp120 reactivity, as determined by endpoint antibody binding titration. (B) Identical guinea pig sera were used in a competition assay with soluble BaL gp120 to determine specificities of sera generated using pseudovirion (VLP) immunizations. Serial dilutions of guinea pig sera were incubated with 200 ng/ml BaL gp120 for 1 h prior to adding HIV-1 BaL virus, which was followed by an additional 1-h incubation period and performance of the neutralization assay described in Materials and Methods. The percent reduction in 50% neutralization titer is indicated in the lower left corner of the respective titration curve set. TMx, TiterMax Gold.
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