Convergence and divergence of B cell responses in two HIV-1 Env immunizations in Rhesus macaques

Abstract

Background: Sequential multivalent immunizations are used to counter diversity in rapidly mutating viruses. Here, we evaluated the effect of HIV-1 immunogen formats on the binding profile of memory B-cells elicited in two independent Rhesus macaque trials.

Methods: In one trial, female Rhesus macaques were immunized with a multiclade HIV-1 gp120 envelope glycoprotein (Env) cocktail and bled two weeks post final immunization. In another trial, male and female Rhesus macaques were sequentially immunized with clonally-related Env glycoproteins: Four immunogens were administered as non-stabilized gp140 Envs and the fifth as a specially stabilized gp140 Env trimer (SOSIP); animals were bled before and after SOSIP immunization. Immunogen-binding peripheral memory B-cells were sorted and cultured at limiting dilution. Culture supernatants were assessed by ELISA for binding to individual immunogens.

Results: In the first trial, 81% (591/734) of B-cells cross-react with multiple Envs and most bind to all immunogens. In the second trial, 81% (331/410) of B-cells isolated before SOSIP administration react with all non-stabilized gp140 Env immunogens and 27% also cross-react with the yet-to-be-administered SOSIP-stabilized Env. However, after SOSIP administration, SOSIP-stabilized trimer-reactive B-cells increase to 86% (219/256) but most (82%) do not cross-react with the preceding immunogens.

Conclusions: Multiclade and sequential regimens before SOSIP-stabilized Env immunization elicited B-cells that converge on shared epitopes. A change in immunogen format results in a divergent B-cell response that vastly fails to engage prior responses. Critically, B-cell priming with non-stabilized Env cannot modify the effect of the epitope immunodominance hierarchy in a SOSIP trimer. These results suggest that a change in immunogen format may cause off-target B-cell engagement, but also that B-cell repriming is possible despite pre-existing immunity.

Fig. 1. Tetravalent gp120 Env immunization.

a NHP immunization scheme with multivalent HIV-1 gp120 Env cocktails delivered as DNA and monomeric proteins. i.d. intradermal, i.m. intramuscular. b Viremia time-course of the four NHPs included in this study post-seroconversion or after the fifteenth challenge for RVv15. Each NHP is color-coded as follows: red circles: RNs14; blue squares: RPt15; orange triangles: RSf15; green inverted triangles: RSf15. Dashed line at assay limit of sensitivity threshold (60 copies/ml). Measurements were performed in technical duplicates. Also see Supplementary Data 1. c Serum was collected four weeks prior to immunization and again at weeks 10 and 18 post-immunization. Peak serum neutralization data, defined as highest ID₅₀ value within the time course, are shown for the four NHPs in this study against two tier 1 viruses (C.MW965.26 and TH023.6) and four tier 2 viruses (A.92UG037.1, B.JR-FL, B.WITO4160.33, and C.Du172.17). Each NHP is color-coded as indicated in panel b. Lines indicate group mean and standard deviation. Measurements were performed in technical duplicates. Also see Supplementary Data 2. d Immunogen binding profiles of 734 B cells isolated from all four NHPs. Reactivity with individual and multiple gp120 Env immunogens is color coded as indicated: yellow: A.92UG037 gp120 only; orange: B.JR-FL gp120 only; red: C.93MW965 gp120 only; brown: AE.consensus gp120 only; light blue: 2 gp120 Envs; violet: 3 gp120 Envs; dark blue: all 4 gp120 Envs. Also see Supplementary Data 3. e Binding to individual immunogens of monoreactive B cells, expressed as percentage of the total number of monoreactive B cells. Data are shown as aggregate (“All NHPs”) and for each NHP. Reactivity with individual and multiple gp120 Env immunogens is color coded as indicated in panel d. Also see Supplementary Data 4. f Immunogen cross-reactivity of multireactive B cells, expressed as percentage of the total number of multireactive B cells, shown as aggregate (“All NHPs”) and for each NHP. Reactivity with individual and multiple gp120 Env immunogens is color coded as indicated in panel d. Also see Supplementary Data 5.

Fig. 2. Sequential immunization with clonally related Envs.

a NHP immunization scheme (see methods and ref. ). TF transmitted founder. b, c Cross-reactivity of B cells with non-stabilized gp140 Envs immunogens (outer ring) and stabilized CH505.w136 SOSIP trimer (inner ring) (b) before and (c) after CH505.w136 SOSIP trimer immunizations. Data are shown as aggregate (“All NHPs”) and for each NHP. Number of B cells analyzed is shown for each chart. Reactivity with individual and multiple non-stabilized and SOSIP stabilized gp140 Env immunogens is color coded as follows: gray: no reactivity with non-stabilized gp140 Envs (0 gp140); yellow: reactivity with one non-stabilized gp140 Env; orange: reactivity with two non-stabilized gp140 Envs; red: reactivity with three non-stabilized gp140 Envs; brown: reactivity with four non-stabilized gp140 Envs; white: no reactivity with SOSIP-stabilized gp140 Env; green: reactivity with SOSIP-stabilized gp140 Env. Also see Supplementary Data 6 and 7.

Fig. 3. Somatic hypermutation frequencies of IgV_H gene segments isolated from memory B cells collected after the end of the sequential immunization with IDLV expressing CH505 Envs.

We analyzed a total of 150 IgV_HDJ_H sequences from memory B cells isolated at week 117 that bound to either all immunogens (n = 20, left column) or to the CH505.wk136 SOSIP-stabilized trimer immunogen only (n = 130, right column). IgV_H gene segment somatic hypermutation frequencies (y-axis) were calculated using Cloanalyst (see methods). Lines show the mean and standard deviation for each group. Statistically significant difference between the two groups (p = 0.0001) was evaluated using the non-parametric, two-tailed Mann-Whitney test. The NHP source for each sequence is coded as follows: red triangles: Rh6575; blue squares: Rh6600; black circles: Rh6601. Also see Supplementary Data 8 and 9.