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Comparative Study
. 2004 Apr;78(8):4029-36.
doi: 10.1128/jvi.78.8.4029-4036.2004.

Selective modification of variable loops alters tropism and enhances immunogenicity of human immunodeficiency virus type 1 envelope

Zhi-yong Yang  1 , Bimal K Chakrabarti, Ling Xu, Brent Welcher, Wing-pui Kong, Kwanyee Leung, Amos Panet, John R Mascola, Gary J Nabel
Affiliations
Comparative Study

Selective modification of variable loops alters tropism and enhances immunogenicity of human immunodeficiency virus type 1 envelope

Zhi-yong Yang et al. J Virol. 2004 Apr.

Erratum in

  • J Virol. 2006 Apr;80(8):4206

Abstract

Although the B clade of human immunodeficiency virus type 1 (HIV-1) envelopes (Env) includes five highly variable regions, each of these domains contains a subset of sequences that remain conserved. The V3 loop has been much studied for its ability to elicit neutralizing antibodies, which are often restricted to a limited number of closely related strains, likely because a large number of antigenic structures are generated from the diverse amino acid sequences in this region. Despite these strain-specific determinants, subregions of V3 are highly conserved, and the effects of different portions of the V3 loop on Env tropism and immunogenicity have not been well delineated. For this report, selective deletions in V3 were introduced by shortening of the stem of the V3 loop. These mutations were explored in combination with deletions of selected V regions. Progressive shortening of the stem of V3 abolished the immunogenicity as well as the functional activity of HIV Env; however, two small deletions on both arms of the V3 stem altered the tropism of the dualtropic 89.6P viral strain so that it infected only CXCR4(+) cells. When this smaller deletion was combined with removal of the V1 and V2 loops and used as an immunogen in guinea pigs, the antisera were able to neutralize multiple independent clade B isolates with a higher potency. These findings suggest that highly conserved subregions within V3 may be relevant targets for eliciting neutralizing antibody responses, affecting HIV tropism, and increasing the immunogenicity of AIDS vaccines.

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Figures

FIG. 1.
FIG. 1.
Schematic representation of Env mutations. (A) The major structural motifs in HIV Env are shown, together with the selected expression vectors used in these studies. V1, V2, V3, and V4 indicate the respective variable regions, and the sequence of the relevant V3 loop (HXB2/BaL chimera) is indicated. (B) Schematic structure of the V3 loop (HXB2/BaL chimera) with V3 (1AB) stem-shortening mutations.
FIG. 2.
FIG. 2.
Mutations in the stem of the V3 loop and protein expression of various gp145ΔCFI (HXB2/BaL chimera) V3 deletion mutants. (A) Sequences of progressive V3 stem deletion mutations in Env from the HXB2/BaL chimera. (B) Protein expression of gp145ΔCFI (HXB2/BaL chimera) V3 deletion mutant expression vectors. The indicated mutations in the gp145ΔCFI constructs, described previously (7), were prepared and analyzed by SDS-PAGE followed by Western blot analysis with human monoclonal antibody 2F5. Plasmid expression vectors encoding the indicated mutants were transfected into 293 cells by use of calcium phosphate. Cell lysates were collected 48 h later. (C) Expression of gp145 (HXB2/BaL chimera) V3 deletion mutant vectors with the V1 and V2 regions deleted.
FIG. 3.
FIG. 3.
Effects of mutations in the stem of the V3 loop on tropism of 89.6P Env. (A) Buoyant density sedimentation analysis of indicated V3 mutants in lentiviral vector particles, performed as described in Materials and Methods. (B) Effects of V3 mutations in strain 89.6P Env on infection of a CXCR4-tropic cell line, MT-2 (left), and a CCR-5-tropic indicator cell line, MAGI-CCR5 (right), using a luciferase reporter gene. The positions of the indicated V3 mutations in strain 89.6P Env are the same as those shown for the HXB2/BaL chimera (Fig. 2A). Both codon-modified and wild-type (wt) 89.6P Envs were used as positive controls.
FIG. 4.
FIG. 4.
Expression of different HIV gp145 (HXB2/BaL chimera) V region mutants and induction of neutralizing antibodies. (A) Expression of the indicated HXB2/BaL V region mutants was determined by SDS-PAGE followed by Western blotting with transfected 293 cells. (B) Neutralizing activity against BaL in sera from guinea pigs immunized with the indicated DNA/Ad expression vectors. Sera were tested at a 1:5 dilution. Results are the means ± standard errors of four guinea pig sera for each construct. The P value shown is the result of a Mann-Whitney test comparing the median neutralization value of the two groups indicated.
FIG. 5.
FIG. 5.
Characterization of antibody response induced by gp145 (HXB2/BaL chimera) ΔV1V2 and selected V3 deletion mutants. (A) Neutralization activity against BaL induced by immunization of guinea pigs with the indicated mutants, including the ΔV1V2 deletion mutants and the ΔV1V2V3(1AB) stem-shortening mutants. Sera were tested at a 1:5 dilution. Results are the means ± standard errors of four guinea pig sera for each construct. Results from one of two independent experiments are shown. The sera tested were independent from the sera tested for Fig. 4B. (B) Total ELISA titers in the same guinea pig sera are shown and are comparable between the different mutants.
FIG. 6.
FIG. 6.
Comparison of breadth and potency of the antibody response induced by selected V region mutants. (A) IC50 titers against four clade B primary isolates are indicated (see Materials and Methods). Results are the means ± standard errors of four guinea pig sera for each construct. The statistically significant differences between ΔV1V2V3(1AB) and the wild-type gp140/145 are shown (Mann-Whitney test). (B) Four individual sera from ΔV1V2V3(1AB)-immunized guinea pigs were screened against a panel of 10 primary viruses. Sera were tested at a 1:5 dilution. Percentages of neutralization (compared with corresponding preimmune sera) are indicated. Data shown are averages of two experiments.
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References

    1. Aoki, K., C. Barker, X. Danthinne, M. J. Imperiale, and G. J. Nabel. 1999. Efficient generation of recombinant adenoviral vectors by Cre-lox recombination in vitro. Mol. Med. 5:224-231. - PMC - PubMed
    1. Briggs, D. R., D. L. Tuttle, J. W. Sleasman, and M. M. Goodenow. 2000. Envelope V3 amino acid sequence predicts HIV-1 phenotype (co-receptor usage and tropism for macrophages). AIDS 14:2937-2939. - PubMed
    1. Bures, R., A. Gaitan, T. Zhu, C. Graziosi, K. M. McGrath, J. Tartaglia, P. Caudrelier, R. El Habib, M. Klein, A. Lazzarin, D. M. Stablein, M. Deers, L. Corey, M. L. Greenberg, D. H. Schwartz, and D. C. Montefiori. 2000. Immunization with recombinant canarypox vectors expressing membrane-anchored glycoprotein 120 followed by glycoprotein 160 boosting fails to generate antibodies that neutralize R5 primary isolates of human immunodeficiency virus type 1. AIDS Res. Hum. Retrovir. 16:2019-2035. - PubMed
    1. Burns, D. P., and R. C. Desrosiers. 1994. Envelope sequence variation, neutralizing antibodies, and primate lentivirus persistence. Curr. Top. Microbiol. Immunol. 188:185-219. - PubMed
    1. Burton, D. R. 2002. Antibodies, viruses and vaccines. Nat. Rev. Immunol. 2:706-713. - PubMed

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