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. 2012 Mar;86(5):2488-500.
doi: 10.1128/JVI.06259-11. Epub 2011 Dec 28.

Targeting HIV-1 envelope glycoprotein trimers to B cells by using APRIL improves antibody responses

Mark Melchers  1 , Ilja Bontjer, Tommy Tong, Nancy P Y Chung, Per Johan Klasse, Dirk Eggink, David C Montefiori, Maurizio Gentile, Andrea Cerutti, William C Olson, Ben Berkhout, James M Binley, John P Moore, Rogier W Sanders
Affiliations

Targeting HIV-1 envelope glycoprotein trimers to B cells by using APRIL improves antibody responses

Mark Melchers et al. J Virol. 2012 Mar.

Abstract

An HIV-1 vaccine remains elusive, in part because various factors limit the quantity and quality of the antibodies raised against the viral envelope glycoprotein complex (Env). We hypothesized that targeting Env vaccines directly to B cells, by fusing them to molecules that bind and activate these cells, would improve Env-specific antibody responses. Therefore, we fused trimeric Env gp140 to A PRoliferation-Inducing Ligand (APRIL), B-cell Activating Factor (BAFF), and CD40 Ligand (CD40L). The Env-APRIL, Env-BAFF, and Env-CD40L gp140 trimers all enhanced the expression of activation-induced cytidine deaminase (AID), the enzyme responsible for inducing somatic hypermutation, antibody affinity maturation, and antibody class switching. They also triggered IgM, IgG, and IgA secretion from human B cells in vitro. The Env-APRIL trimers induced higher anti-Env antibody responses in rabbits, including neutralizing antibodies against tier 1 viruses. The enhanced Env-specific responses were not associated with a general increase in total plasma antibody concentrations, indicating that the effect of APRIL was specific for Env. All the rabbit sera raised against gp140 trimers, irrespective of the presence of CD40L, BAFF, or APRIL, recognized trimeric Env efficiently, whereas sera raised against gp120 monomers did not. The levels of trimer-binding and virus-neutralizing antibodies were strongly correlated, suggesting that gp140 trimers are superior to gp120 monomers as immunogens. Targeting and activating B cells with a trimeric HIV-1 Env-APRIL fusion protein may therefore improve the induction of humoral immunity against HIV-1.

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Figures

Fig 1
Fig 1
Chimeric Env-APRIL, Env-BAFF, and Env-CD40L proteins are properly folded. (A) Schematic of the Env, Env-APRIL, Env-BAFF, and Env-CD40L proteins. The clade B JR-FL gp140 protein (amino acids 31 to 681) contains several modifications for stabilization that have been previously described (see Materials and Methods). Codon-optimized sequences encoding the active domains of human and rabbit APRIL, BAFF, or CD40L were added to the gp140 open reading frame at the C terminus. (B) Reducing SDS-PAGE and (C) BN-PAGE analysis of Env, Env-APRIL, Env-BAFF, and Env-CD40L proteins secreted from transiently transfected 293T cells. Recombinant purified JR-FL gp120 (50 ng) and Env from a control transfection were included for comparison. (D) Recognition of Env-APRIL and Env-BAFF by MAbs and CD4. The Env-APRIL and Env-BAFF proteins were immunoprecipitated by CD4-IgG2 and HIVIg MAbs to gp120 (b12) or gp41 (2F5) or via a CD4-induced epitope (MAb 17b) on gp120 in the absence or presence of soluble CD4 (sCD4), followed by reducing SDS-PAGE and Western blot analysis.
Fig 2
Fig 2
Env, Env-APRIL, Env-BAFF, and Env-CD40L induce AID expression and Ig secretion in human B cells. (A) AID mRNA levels in naïve human B cells stimulated with Env, Env-fusion proteins, or control stimuli were measured by real-time PCR. Negative-control B cells did not receive any stimulus (no IL-4 or IL-10). Positive-control cells received CpG-B in addition to the stimulation cocktail. Similar results were obtained with naïve B cells from three different donors. (B) IgM (left panel), IgG (middle panel), and IgA (right panel) levels in the supernatant of human B cells mock stimulated or stimulated with Env or the respective fusion proteins supplemented with a maturation cocktail were measured by ELISA after 14 days. Similar results were obtained with B cells from three different donors. *, **, and *** indicate P < 0.05, P < 0.01, and P < 0.001, respectively, as determined by a one-tailed Student's t test.
Fig 3
Fig 3
Env-APRIL induces higher antibody titers than Env in rabbits. (A) Rabbit immunization scheme. (B) Midpoint anti-gp120 IgG titers over the course of the immunization experiment as determined by ELISA. The mean values from 4 rabbits per group are given. (C) Midpoint anti-gp120 IgG titers at week 18. The mean values ± standard errors of the means (SEM) derived from 4 rabbits are shown. (D) Fold induction of IgG anti-gp120 titers. The mean relative increases ± SEM of the results from 4 rabbits at week 18 compared to week 16 are given. (E) Total IgG midpoint titers at weeks 0, 12, and 18. Mean values ± SEM are given. * indicates P < 0.05 as determined by a one-tailed Mann-Whitney test.
Fig 4
Fig 4
nAb responses mature over time. (A) The mean 50% SF162 neutralization titers are shown for each group. (B) The ratios of the 50% SF162 neutralization titers and the midpoint anti-gp120 titers at weeks 6, 12, and 18 are plotted for each rabbit. ** and *** indicate P < 0.01 and P < 0.001, respectively, as determined by a one-tailed Mann-Whitney test.
Fig 5
Fig 5
nAb titers correlate with anti-trimer binding antibody titers. (A) Mean midpoint trimer-binding titers ± SEM as determined by ELISA at weeks 0, 12, and 18. * indicates P < 0.05. (B to E) The 50% SF162 neutralization titers of each individual rabbit (week 12 and 18) are plotted against the midpoint anti-gp120 binding antibody titer (B) and the midpoint anti-trimer binding antibody titer (C). (D and E) Data were determined as described for panels B and C, but only 50% JR-FL neutralization titers are shown. (F) Ratios of the midpoint anti-trimer/anti-gp120 binding antibody titers at weeks 0, 12, and 18. Symbols are the same as described for Fig. 4. ** indicates P < 0.01 as determined by a one-tailed Mann-Whitney test. (G to I) Midpoint neutralization titers for MN (G), SF162.LS (H), and BaL.26 (I) are plotted against the ratio of the midpoint anti-trimer/anti-gp120 binding antibody titers. The Spearman r-values and two-tailed P values are shown.
Fig 6
Fig 6
Sera from gp140-immunized animals recognize native trimers. The binding of representative sera from each group to native JR-FL Env trimers (A) and JR-FL D368R mutant trimers (B) was examined. nAbs b12 and 2F5 and nonneutralizing antibody 15e served as controls. A quantitative evaluation of the trimer band intensity is presented in the bar graphs, where small bars represent efficient trimer binding of the sera and a resulting decrease in the intensity of the trimer band.
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