Diverse recombinant HIV-1 Envs fail to activate B cells expressing the germline B cell receptors of the broadly neutralizing anti-HIV-1 antibodies PG9 and 447-52D

Abstract

Broadly neutralizing antibodies (bNAbs) against HIV-1 are generated during HIV-1-infection but have not yet been elicited by immunization with recombinant forms of the viral envelope glycoprotein (Env; the target of anti-HIV-1 neutralizing antibodies). A particular type of bNAb targets the CD4-binding site (CD4-BS) region of Env. These antibodies are derived from a limited number of VH/VL genes and can bind to and neutralize diverse HIV-1 strains. Recent reports have demonstrated the limited potential of Env to activate B cells expressing the germline B cell receptor (BCR) forms of anti-CD4-BS bNAbs. A potential reason for the lack of elicitation of anti-CD4-BS bNAbs by Env immunogens is the absence of stimulation of naive B cells expressing the germline BCRs of such antibodies. Several bNAbs have been isolated from HIV-1-infected subjects that target other structurally conserved regions of Env. How frequently Env immunogens stimulate the germline BCRs that give rise to bNAbs that target Env regions other than the CD4-BS is not well understood. Here, we investigated the interactions between diverse Envs and the BCRs of known bNAbs targeting not only the CD4-BS but also conserved elements of the second and third variable Env regions. Our results indicate that Env is generally ineffective in engaging germline BCRs of bNAbs irrespective of their epitope target. Potentially, this is the result of viral evolutionary mechanisms adopted to escape broadly neutralizing antibody responses. Our results also suggest that a single Env capable of activating germline BCRs that target distinct Env epitopes will be very difficult to identify or to design.

Importance: Broadly neutralizing antibodies against HIV-1 are thought to be an important component of the immune responses that a successful vaccine should elicit. Broadly neutralizing antibodies are generated by a subset of those infected by HIV-1, but so far, they have not been generated by immunization with recombinant Envelope (Env, the target of anti-HIV-1 neutralizing antibodies). Here, we provide evidence that the inability of Env to elicit the production of broadly neutralizing antibodies is due to the inability of diverse Envs to engage the germline B cell receptor forms of known broadly neutralizing antibodies.

FIG 1

Env-mediated B cell activation through the mutated but not the predicted germline b12 BCR. (A) Binding of mutated b12 IgG to the indicated gp140 trimers measured by ELISA. Abs 450 nm, absorbance at 450 nm. (B) B cells expressing either the mutated or the predicted germline version of the b12 BCR, as well as mock-transfected cells, were incubated with the indicated gp140 trimers. Their binding was determined as described in Materials and Methods. (C) Ca²⁺ flux response in B cells expressing the mutated b12 BCR incubated with the indicated trimeric gp140 proteins. The black arrow indicates the time of trimer addition. (D) The area under the curve of the Ca²⁺ flux response (determined in panel C) through the mutated b12 BCR correlates with the affinity of soluble b12 IgG to SF162, QHO692, 405c, 426c, and 706c gp140 proteins (determined in panel A). (E) Ca²⁺ flux response in B cells expressing the predicted germline b12 BCR to the indicated trimeric gp140 proteins. The black arrow indicates the time of trimer addition.

FIG 2

Env-mediated B cell activation through the mutated but not the predicted germline PG9 BCRs. (A) Binding of mutated PG9 IgG to the indicated gp140 trimers measured by ELISA. (B) Binding of the indicated gp140 trimeric Envs to B cells expressing either the mutated or the predicted germline PG9 BCR, as well as mock-transfected cells. (C) Ca²⁺ flux response in B cells expressing mutated PG9 BCR following incubation with the indicated trimeric gp140 proteins. The black arrow indicates the time of Env addition. (D) Ca²⁺ flux response in B cells expressing the predicted germline PG9 BCR following incubation with the indicated trimeric gp140 proteins. The black arrow indicates the time of Env addition.

FIG 3

Env-mediated B cell activation through the mutated but not the predicted germline 447-52D BCR. (A) Binding of the mutated 447-52D IgG to the indicated gp140 trimers as measured by ELISA. (B) Binding of the indicated gp140 trimeric Envs to B cells expressing either the mutated or predicted germline version of the 447-52D BCR. Results with mock transfected cells are also included. (C) Ca²⁺ flux response in B cells expressing mutated 447-52D BCR incubated with the indicated trimeric gp140 proteins. The black arrow indicates the time of Env addition. (D) The area under the curve of the Ca²⁺ flux response (determined in panel C) through the mutated 447-52D BCR correlates with the affinity of sIgG 447-52D (determined in panel A). (E) Ca²⁺ flux response in B cells expressing the predicted germline 447-52D BCR following incubation with the indicated trimeric gp140 proteins. The black arrow indicates the time of Env addition.

FIG 4

Selective disruption of NLGS on Env does not confer binding to germline b12. Binding of mutated and predicted germline b12, mutated and predicted germline NIH45-46, and polyclonal HIV IgG to trimeric 426c gp140 (A), trimeric 426c containing mutations disrupting NLGS in the V5 and D loop regions (B), trimeric QH0692 gp140 (C), and trimeric QH0692 containing mutations disrupting NLGS in the V5 and D loop regions (D). Ca²⁺ flux in B cells expressing the mutated (E) or the predicted (F) germline b12 BCR in response to the trimeric wild-type and NLGS-mutated 426c and QH0692 (426c.N276D.N460D.N463D and QH0692.N276D.N462D, respectively) as well as the SF162-D368R trimer. Black arrows indicate the time of Env addition.

FIG 5

Selective disruption of NLGS on Env ablates binding to the predicted germline 447-52D. Binding to 1 μM solutions of trimeric SF162, QH0692, QH0692.T297A.T303A.S334A, and SF162-ΔV3 by mutated 447-52D (A) and predicted 447-52D (B) germline sIgGs measured by BLI.

FIG 6

HA stimulates B cells expressing germline BCRs of anti-influenza bNAbs. B cells expressing the indicated mutated or predicted germline BCRs are stimulated with 30 μg/ml recombinant trimeric HA (H5/VN/04) (A) or 30 μg/ml recombinant trimeric SF162-K160N (B). Black arrows indicate the time of HA and Env addition.