Targeted selection of HIV-specific antibody mutations by engineering B cell maturation

Abstract

INTRODUCTION: A major goal of HIV-1 vaccine development is the design of immunogens that induce broadly neutralizing antibodies (bnAbs). However, vaccination of humans has not resulted in the induction of affinity-matured and potent HIV-1 bnAbs. To devise effective vaccine strategies, we previously determined the maturation pathway of select HIV-1 bnAbs from acute infection through neutralizing antibody development. During their evolution, bnAbs acquire an abundance of improbable amino acid substitutions as a result of nucleotide mutations at variable region sequences rarely targeted by activation-induced cytidine deaminase, the enzyme responsible for antibody mutation. A subset of improbable mutations is essential for broad neutralization activity, and their acquisition represents a key roadblock to bnAb development.

RATIONALE: Current bnAb lineage-based vaccine strategies can initiate bnAb lineage development in animal models but have not specifically elicited the improbable mutations required for neutralization breadth. Induction of bnAbs requires vaccine strategies that specifically engage bnAb precursors and subsequently select for improbable mutations required for broadly neutralizing activity. We hypothesized that vaccination with immunogens that bind with moderate to high affinity to bnAb B cell precursors, and with higher affinity to precursors that have acquired improbable mutations, could initiate bnAb B cell lineages and select for key improbable mutations required for bnAb development.

RESULTS: We elicited serum neutralizing HIV-1 antibodies in human bnAb precursor knock-in mice and wild-type macaques vaccinated with immunogens designed to select for improbable mutations. We designed two HIV-1 envelope immunogens that bound precursor B cells of either a CD4 binding site or V3-glycan bnAb lineage. In vitro, these immunogens bound more strongly to bnAb precursors once the precursor acquired the desired improbable mutations. Vaccination of macaques with the CD4 binding site–targeting immunogen induced CD4 binding site serum neutralizing antibodies. Antibody sequences elicited in human bnAb precursor knock-in mice encoded functional improbable mutations critical for bnAb development. In bnAb precursor knock-in mice, we isolated a vaccine-elicited monoclonal antibody bearing functional improbable mutations that was capable of neutralizing multiple HIV-1 global isolates. Structures of a bnAb precursor, a bnAb, and the vaccine-elicited antibody revealed the precise roles that acquired improbable mutations played in recognizing the HIV-1 envelope. Thus, our immunogens elicited antibody responses in macaques and knock-in mice that exhibited the mutational patterns, structural characteristics, or neutralization profiles of nascent broadly neutralizing antibodies.

CONCLUSION: Our study represents a proof of concept for targeted selection of improbable mutations to guide antibody affinity maturation. Moreover, this study demonstrates a rational strategy for sequential immunogen design to circumvent the difficult roadblocks in HIV-1 bnAb induction by vaccination. We show that immunogens should exhibit differences in affinity across antibody maturation stages where improbable mutations are necessary for the desired antibody function. This strategy of selection of specific antibody nucleotides by immunogen design can be applied to B cell lineages targeting other pathogens where guided affinity maturation is needed for a protective antibody response.

Fig. 1.. Structural characterization and development of an HIV-1 Env antigen capable of binding to the DH270 unmutated common ancestor (UCA).

(A) Antibody neutralization of TZM-bl cell infection was compared for HIV-1 pseudoviruses encoding CH848 10.17 gp160 Env or the same Env with glycosylation sites at Asn¹³³ and Asn¹³⁸ (N133 and N138) removed by mutating them to Asp¹³³ and Thr¹³⁸ (10.17DT). Neutralization titers are shown as IC₅₀ values. (B) The binding affinities of DH270 UCA and the first intermediate antibody in the DH270 lineage (IA4) for CH848 10.17DT–stabilized Env trimers was determined by surface plasmon resonance. The somatic mutations acquired in the heavy chain variable region (V_H) and light chain variable region (V_L) of DH270 IA4 are shown above and below the arrow, respectively. Improbable and probable somatic mutations were modeled on the DH270 UCA Fab crystal structure (PDB ID 5U0R) as red and green residues, respectively. (C) CH848 10.17DT calcium flux induction in DH270 UCA (green), DH270.1 (orange), and negative control (black) IgG-expressing Ramos B cells. (D) Cryo-EM reconstruction (4.2 Å resolution) of DH270 UCA (green) bound to CH848 10.17DT-stabilized Env trimers (gray), segmented by components. (E) Zoomed-in view of the interactive region between DH270 UCA and CH848 10.17DT. HIV-1 Env gp120 is shown in gray, with the V3 loop in orange and the V1 loop in red. Glycans are shown in stick representation and colored by elements, with oxygen atoms in red and nitrogen atoms in blue. The heavy chain of DH270 UCA is shown in green and light chain in yellow. Color code for complementarity-determining regions (CDRs) of the antibody: wheat, CDR H1; blue, CDR H2; purple, CDR H3; brown, CDR L1; pink, CDR L2; teal, CDR L3. DH270 UCA contacted the V1 loop, the V3 loop, and the surrounding glycans, with contacts made by both the heavy and light chains of the antibody. (F to H) Zoomed-in views of the structure showing details of the contacts DH270 UCA makes with glycan 332, shown in stick representation overlaid with dots and colored by element, with the DH270 UCA shown in surface representation (F), the V3 loop (G), and the V1 loop (H).

Fig. 2.. CH848 10.17DT nanoparticle immunization of DH270 UCA knock-in mice induces serum neutralizing antibody responses and the improbable G57R mutation.

(A) Negative-stain electron microscopy of CH848 10.17DT–stabilized Env trimers conjugated to ferritin nanoparticles. A magnified image of the Env nanoparticles (top) and 2D class average (bottom) of the Env nanoparticles are shown at the right. (B) Antigenic profile of CH848 10.17DT–stabilized Env trimers conjugated to ferritin nanoparticles or unconjugated. Binding was determined by biolayer interferometry and normalized to PGT151 binding values. The normalized binding is shown in the heat map, with a value of 1 being equal to PGT151 binding. OD glycan, outer domain glycans in the high-mannose patch. (C) CH848 10.17DT Env trimer nanoparticle (red) induction of calcium flux in homozygous (V_H^+/+, V_L^+/+) DH270 UCA double knock-in (double KI) mouse B cells. (D) Heterozygous (V_H^+/−, V_L^+/−) DH270 UCA double KI mouse immunization regimen with CH848 10.17DT SOSIP trimer nanoparticles. (E) Quantification of the frequency of splenic germinal center B cells (top) and follicular helper T cells (bottom) in vaccinated DH270 UCA mice. Unvaccinated, wild-type C57BL/6 mice (BL6) served as baseline controls. Means ± SEM are shown by black bars. *P < 0.05 (Wilcoxon exact test). (F) Serum antibody neutralization of autologous tier 2 CH848 10.17DT (top) and CH848 10.17 (bottom) virus infection of TZM-bl cells. Group geometric mean ID₅₀ titers are shown (nanoparticle, n = 5; adjuvant only, n = 6). Arrows indicate immunization time points. (G) Top: Enumeration by V_H next-generation sequencing of the frequency of unique DH270 sequences encoding the G57R amino acid change. Frequencies of G57R were determined in total splenocytes 1 week after the sixth immunization. Each mouse is shown by an individual symbol; horizontal bars indicate the group mean. Bottom: Relative (fold) increase in G57R frequency in heterozygous DH270 UCA KI mice immunized with CH848 10.17DT Env nanoparticle and adjuvant compared to adjuvant-only control mice. Box-and-whisker plots indicate the minimum, maximum, median, and interquartile range fold increase for each group. Each symbol represents an individual mouse. *P < 0.05 (Wilcoxon exact test).

Fig. 3.. CH848 10.17DT Env trimer nanoparticle immunization elicits neutralizing antibody DH270.mu1 that encodes both S27Y and G57R improbable amino acid changes.

(A) The percentage of CH848 10.17–specific B cells sorted from CH848 10.17DT Env trimer nanoparticle–immunized mice that encode DH270 V_H and V_L regions. CH848 10.17–specific B cells encoding one or more mouse Ig chains were classified as other antibodies. Total number of antibodies isolated is shown in the center of the pie chart. (B) The percentage of DH270-expressing B cells that have acquired one or more somatic mutations. Total number of DH270 antibodies isolated [based on data in (A)] is shown in the center of the pie chart. (C) Amino acid mutation frequency of the DH270 V_H and V_L regions cloned from the sorted CH848 10.17–specific B cells shown in (B). Box-and-whisker plots show the range, median, and interquartile range. (D) Env nanoparticle vaccine–induced monoclonal antibody DH270.mu1 possessed both the G57R and S27Y amino acid changes in its HCDR2 and LCDR1 loops, respectively. Shown is an amino acid alignment of select DH270 clonal lineage antibodies and DH270.mu1. The two critical improbable mutations are highlighted in yellow. (E) DH270.mu1 neutralizes autologous and heterologous viruses with potency nearly identical to that of DH270 IA4. Neutralization titers are shown as IC₅₀ for each individual virus. Left, vaccine-induced antibody; right, DH270 intermediate antibody inferred from the clonal lineage elicited during chronic HIV-1 infections. (F) DH270.mu1 possesses neutralization breadth (16/23 or 70% of viruses) similar to that of DH270 IA4. Neutralization IC₅₀ titers are shown in a heat map. (G) The somatic mutations shared between the vaccine-elicited antibodies and the bnAb DH270.1. The DH270.1 somatic mutations observed after vaccination with 10.17DT trimer nanoparticle are indicated with blue squares; unobserved mutations are shown by white squares. (H) The frequency of the total (n = 134) mouse mAbs isolated from mice immunized with the 10.17DT SOSIP nanoparticle that had the indicated numbers of shared mutations with DH270.1. Amino acid abbreviations: A, Ala; D, Asp; F, Phe; G, Gly; I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val; W, Trp; Y, Tyr.

Fig. 4.. Vaccine-induced antibody DH270.mu1 with improbable mutations binds to the HIV-1 Env SOSIP trimer with the same binding mode as DH270 bnAbs.

(A) Cryo-EM reconstruction (4.3 Å resolution) of DH270.6 (cyan) bound to CH848 10.17DT-stabilized Env trimers (gray) segmented by components. (B) Same as (A) but for the DH270.mu1 complex (3.5 Å resolution) with DH270.mu1 shown in pink. (C) Zoomed-in view of the interactive region between DH270.6 and CH848 10.17DT. HIV-1 Env gp120 is shown in gray with the V3 loop in orange and the V1 loop in red. Glycans are shown in stick representaron and colored by elements, with oxygen atoms in red and nitrogen atoms in blue. The heavy chain of DH270.6 is shown in cyan and light chain in yellow. Color code for CDRs: wheat, CDR H1; blue, CDR H2; purple, CDR H3; brown, CDR L1; pink, CDR L2; teal, CDR L3. (D) Same as (C) but for the DH270.mu1 complex, with DH270.mu1 heavy chain in pink. (E) Overlay of the DH270-bound V1 (green), DH270.6-bound V1 (blue), and DH270.mu1-bound V1 (magenta). (F to H) Zoomed-in views showing details of the contacts made by heavy chain residue 57. Black dashed lines indicate hydrogen bonds. (I to K) Zoomed-in views showing details of the contacts made by light chain residue 27.

Fig. 5.. Env trimer immunization elicits neutralizing antibodies with the criticai, improbable K19T amino acid change in the CD4 binding site bnAb lineage CH235.

(A) CH235 UCA neutralization of autologous CH505 virus infection of TZM-bl cells is enhanced by the acquisition of the K19T somatic mutation in the V_H. Neutralization titers are shown as IC₅₀ values. A mixture of CH01 and CH31 was used as a positive control. (B) CH235 UCA (het/het) KI mouse (n = 5) immunization comparing Man₅GlcNAc₂-enriched versus heterogeneously glycosylated CH505 M5.G458Y SOSIP gp140 trimer. (C) Comparable titers of serum antibody neutralization of CH505 M5.G458Y virus infection of TZM-bl cells were elicited by Man₅GlcNAc₂-enriched versus heterogeneously glycosylated CH505 M5.G458Y SOSIP gp140 trimer. Neutralization activity was sensitive to a N280D amino acid change in the CD4 binding site. Neutralization titers are shown as ID₅₀ with each symbol representing one mouse serum sample collected 1 week after the fourth immunization. Group geometric means are shown by horizontal bars. Murine leukemia virus was negative at each time point. (D) Enumeration by V_H next-generation sequencing of the frequency of unique CH235 sequences encoding the K19T amino acid change in mice immunized with M5 gp120 or Man₅GlcNAc₂-enriched M5.G458Y trimer. Frequencies of K19T were determined in total splenocytes 1 week after the final immunization. Each mouse is shown by an individual symbol; horizontal bars indicate the group mean. *P < 0.05 (Wilcoxon exact test); ns, not significant. (E) Fold increase in K19T frequency in heterozygous CH235 UCA KI mice immunized with Man₅GlcNAc₂-enriched M5. G458Y trimer and M5 gp120. Symbols and bars are the same as in (D). *P < 0.05 (Wilcoxon exact test). (F) M5.G458Y gp120-reactive single splenic B cells from a Man₅GlcNAc₂-enriched CH505 M5.G458Y Env trimer–immunized mouse were sorted by fluorescence-activated cell sorting (FACS) 1 week after the final immunization. All of the 57 recovered antibody sequences originated from the CH235 KI variable regions. The pie chart shows the percentage of CH235-expressing B cells that have acquired one or more somatic mutations. (G) Amino acid alignment of V_H sequences from vaccine-induced and infection-induced CH235 antibodies shows the occurrence of the K19T mutation. (H) Vaccine-induced CH235 antibodies encoding K19T neutralize autologous CH505 virus infection of TZM-bl cells more potently than does the CH235 UCA. Viruses were grown under conditions that result in Man₅GlcNAc₂ enrichment (left) or heterogeneous glycosylation (right). The heat maps depict IC₅₀ neutralization titers for each individual virus.

Fig. 6.. CH505 M5.G458Y SOSIP nanoparticle immunization of rhesus macaques elicits serum autologous neutralizing antibodies against the CD4 binding site.

(A) Two-dimensional class averages of negative-stain electron microscopy images of GnTI^−/− cell–expressed M5.G458Y-stabilized SOSIP gp140 ferritin nanoparticles. (B) Rhesus macaque immunization with M5.G458Y-stabilized SOSIP gp140 ferritin nanoparticles enriched for Man₅GlcNAc₂ glycans. (C) Plasma IgG binding over time to GnTI^−/−cell-expressed M5.G458Y-stabilized SOSIP gp140. (D) Serum neutralization kinetics against GnTI^−/− cell–produced HIV-1 CH505 M5.G458Y virus. Each curve represents an individual macaque. (E) Macaque serum after three immunizations neutralizes Man₅GlcNAc₂-enriched and heterogeneously glycosylated CH505 M5.G458Y virus. Horizontal bars are the geometric mean for the group (n = 4). SVA is a negative control murine leukemia virus. (F) Macaque serum after three immunizations neutralizes Man₅GlcNAc₂-enriched CH505 M5.G458Y, M5, and TF viruses. Horizontal bars are the geometric mean for the group (n = 4). (G) Geometric mean of the neutralization titers of all four animals against CH505 M5.G458Y wild-type and CD4bs CH235 bnAb knockout (N280D) viruses over time. (H) Individual macaque neutralization titers after three immunizations against M5.G458Y wild-type and CD4bs CH235 bnAb knockout (N280D) viruses. Horizontal bars are the geometric mean for the group (n = 4). (I) Fold decrease in week 10 neutralization ID₅₀ titers shown in (H) for each macaque upon mutation of the CD4 binding site with a N280D mutation.