An HIV-1 envelope immunogen with W427S mutation in CD4 binding site induced more T follicular helper memory cells and reduced non-specific antibody responses

Abstract

The CD4 binding site (CD4BS) of the HIV-1 envelope glycoprotein (Env) contains epitopes for broadly neutralizing antibody (nAb) and is the target for the vaccine development. However, the CD4BS core including residues 425-430 overlaps the B cell superantigen site and may be related to B cell exhaustion in HIV-1 infection. Furthermore, production of nAb and high-affinity plasma cells needs germinal center reaction and the help of T follicular helper (Tfh) cells. We believe that strengthening the ability of Env CD4BS in inducing Tfh response and decreasing the effects of the superantigen are the strategies for eliciting nAb and development of HIV-1 vaccine. We constructed a gp120 mutant W427S of an HIV-1 primary R5 strain and examined its ability in the elicitation of Ab and the production of Tfh by immunization of BALB/c mice. We found that the trimeric wild-type gp120 can induce more non-specific antibody-secreting plasma cells, higher serum IgG secretion, and more Tfh cells by splenocyte. The modified W427S gp120 elicits higher levels of specific binding antibodies as well as nAbs though it produces less Tfh cells. Furthermore, higher Tfh cell frequency does not correlate to the specific binding Abs or nAbs indicating that the wild-type gp120 induced some non-specific Tfh that did not contribute to the production of specific Abs. This gp120 mutant led to more memory Tfh production, especially, the effector memory Tfh cells. Taken together, W427S gp120 could induce higher level of specific binding and neutralizing Ab production that may be associated with the reduction of non-specific Tfh but strengthening of the memory Tfh.

Figure 1. B cell responses after the immunization by HIV-1 Env gp120 and W427S mutant.

(A–B) Specific binding antibodies in immunized mouse sera to Bal gp120 (A) and HIV-1 06044 gp120 (B) were detected by ELISA and shown as OD450 values at reciprocal serum dilutions. (C–E) HIV-1 Env-specific antibody-secreting cells (ASCs) and total IgG-secreting cells (SCs) were detected by B cell ELISPOT assay. The BM cells were isolated at day 14 after final immunization and added as duplicate in 96-well plate. Biotin-labeled HIV-1 Bal gp120 protein and biotin-labeled anti-mouse IgG antibodies were used for detection of specific ASC and total IgG SCs, respectively. (F) Total IgG levels in immunized mouse sera were measured by quantitative ELISA. WT, wild-type gp120. Data were shown as mean ± SEM. Asterisks indicate statistical significance (* P<0.05, *** P<0.001).

Figure 2. Neutralizing activities of immunized mouse sera.

Neutralization assays were performed against homologous strain 06044 wild-type (A) and SF162 (B) Env-pseudoviruses. The results were expressed as neutralization percentage and the lines of mean neutralization percentage in each group were shown. Asterisks indicate statistical significance (* P <0.05).

Figure 3. The frequency and function of Tfh cells after immunization.

(A–C) Flow cytometry contour plots of splenocytes obtained at day 14 after final immunization (A) and numbers adjacent to outlined areas indicated the frequency (%) CXCR5^hiPD-1^hi cells of total splenic lymphocytes (B) or CD4 T cell (C). (D) The secretory levels of IL-21 in the culture supernatants of splenocytes from immunized mice were detected by quantitative ELISA. (E) Correlation between Tfh frequency and endpoint titer of serum binding antibody. (F) Correlation between Tfh frequency and neutralization percentage. (G) Correlation between the secretory levels of IL-21 and endpoint titer of serum binding antibody. (H) Correlation between the secretory levels of IL-21 and neutralization percentage. R, correlation coefficient. Data were shown as mean ± SEM. WT depict the mice from 06044 WT group and M depict the mice from W427S group.

Figure 4. The specific proliferation activity of splenocytes following immunization.

Splenocytes were collected at day 14 post-final immunization and cultured with 06044 wild-type homologous Env peptide for 4 days. (A) The peptide-specific proliferation was analyzed by CCK-8 assay and results were shown as proliferation index (OD450 values of the splenocytes with the peptide stimulation/OD450 values of cell control). (B) Correlation between the proliferation index and Tfh frequency. (C) Correlation between the proliferation index and serum total IgG concentrations was shown. R, correlation coefficient. Data were shown as mean ± SEM. Asterisks indicate statistical significance (* P<0.05). WT depict the mice from 06044 WT group and M depict the mice from W427S group.

Figure 5. The frequency of memory Tfh (m Tfh) cells.

(A, B) Flow cytometry contour plots of splenocytes obtained at day 14 after final immunization (A), and the frequencies (%) of m Tfh (CD4⁺CXCR5^hiCD44⁺), effector memory Tfh (em Tfh, CD4⁺CXCR5^hiCD44⁺CD62L^-) and central memory Tfh (cm Tfh, CD4⁺CXCR5^hiCD44⁺CD62L⁺) in the CD4⁺ T cell population were analyzed (B). Correlations of endpoint titer (C), neutralization percentage (D) and serum total IgG (E) with frequencies of m Tfh, em Tfh or cm Tfh were analyzed. R, correlation coefficient. Data were shown as mean ± SEM. Asterisks indicate statistical significance (* P<0.05). WT depict the mice from 06044 WT group and M depict the mice from W427S group.