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Antigen pressure from two founder viruses induces multiple insertions at a single antibody position to generate broadly neutralizing HIV antibodies [1]

['Collin Joyce', 'Department Of Immunology', 'Microbiology', 'The Scripps Research Institute', 'La Jolla', 'California', 'United States Of America', 'Consortium For Hiv Aids Vaccine Development', 'Chavd', 'Iavi Neutralizing Antibody Center']

Date: 2023-08

Vaccination strategies aimed at maturing broadly neutralizing antibodies (bnAbs) from naïve precursors are hindered by unusual features that characterize these Abs, including insertions and deletions (indels). Longitudinal studies of natural HIV infection cases shed light on the complex processes underlying bnAb development and have suggested a role for superinfection as a potential enhancer of neutralization breadth. Here we describe the development of a potent bnAb lineage that was elicited by two founder viruses to inform vaccine design. The V3-glycan targeting bnAb lineage (PC39-1) was isolated from subtype C-infected IAVI Protocol C elite neutralizer, donor PC39, and is defined by the presence of multiple independent insertions in CDRH1 that range from 1-11 amino acids in length. Memory B cell members of this lineage are predominantly atypical in phenotype yet also span the class-switched and antibody-secreting cell compartments. Development of neutralization breadth occurred concomitantly with extensive recombination between founder viruses before each virus separated into two distinct population “arms” that evolved independently to escape the PC39-1 lineage. Ab crystal structures show an extended CDRH1 that can help stabilize the CDRH3. Overall, these findings suggest that early exposure of the humoral system to multiple related Env molecules could promote the induction of bnAbs by focusing Ab responses to conserved epitopes.

Broadly neutralizing antibodies (bnAbs) are capable of neutralizing multiple strains of a given virus. Reliable vaccine elicitation of bnAbs represents the most promising solution to stopping the HIV-1 pandemic. BnAbs bind to conserved regions of the HIV envelope glycoprotein (Env) and are typically isolated from infected individuals. Accordingly, an understanding of the complex processes underlying bnAb development may shed light on vaccine design. Here we studied the case of an individual living with HIV-1 who was infected with two similar related founder viruses and went on to mount a broadly neutralizing immune response after 9–11 months of infection. From this individual, we isolated a potent lineage of bnAbs that features different lengths of genetic insertions within a single position. By characterizing the antibody-virus co-evolution that occurs within this individual, we offer insight into how a bnAb response might be facilitated through vaccination.

Funding: This work was supported by the National Institute of Allergy and Infectious Diseases ((U19AI090970 to PP); Consortium for HIV/AIDS Vaccine Development, (UM1AI144462 to B.B., I.A.W., D.R.B.); Center for Viral Systems Biology, (U19AI135995 to B.B.)), the International AIDS Vaccine Initiative (IAVI) through the Neutralizing Antibody Consortium, (SFP1849 to I.A.W, D.R.B.), the Ragon Institute of MGH, MIT and Harvard (D.R.B.) and the James B. Pendleton Charitable Trust (D.R.B.). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Data Availability: Individual Ab HC and LC sequences are available from GenBank database (accession numbers: OQ747680 - OQ747752 and OQ747575 - OQ747679). Individual full-length envelope gene sequences are available from GenBank database (accession numbers: OQ747368 - OQ747574). Raw Illumina and PacBio fastq files are available on the SRA database (BioProject PRJNA943240). Gene expression data is available on the GEO repository (accession number: GSE229232). Fab X-ray crystal structures are available on the PDB (accession numbers: 8GBV, 8GBW, 8GBX, 8GBY, 8GBZ, 8GC0 and 8GC1).

In this study, we investigated a donor who developed a family of potent HIV bnAbs with different lengths of an insertion in a critical framework region following infection with two founder viruses that were closely related and underwent extensive recombination. We used paired heavy-light chain isolation to generate antibodies at different phases of infection and coupled this with sequence studies on viral evolution and structural studies on the antibodies. We conclude that simultaneous exposure of the humoral system to multiple closely related Envs relatively early in infection may assist in the development of neutralization breadth.

Longitudinal studies of bnAb development in HIV infection have highlighted the potential role of superinfection, which can be envisaged to enhance neutralization breadth by focusing antibody responses on epitopes conserved between the infecting and superinfecting viruses. Several studies have indeed suggested that superinfection broadens nAb responses [ 25 , 38 , 39 ], but other studies have not seen such an effect [ 40 , 41 ]. One superinfected donor (CAP256) developed extremely potent bnAbs [ 18 ] but this was shown not to be a property of superinfection directing responses to epitopes conserved between infecting and superinfecting viruses but to a specific response to the superinfecting strain. Another superinfection study reported a similar result [ 42 ]. A further recent study associated the development of neutralization breadth with exposure to multiple closely related founder viruses [ 43 ]. Finally, one of the key elements in the development of many bnAbs is the incorporation of insertions and/or deletions (indels) into antibody sequences [ 44 – 46 ], which is typically viewed as a significant restriction on the induction of bnAbs through vaccination [ 13 ].

Broadly neutralizing antibodies (bnAbs), i.e. those capable of neutralizing multiple strains of a given virus and typically isolated from infected individuals, have attracted much attention as prophylactic and therapeutic agents and as guides for vaccine design [ 1 – 9 ]. BnAbs are crucial in the design of vaccines for highly antigenically variable viruses such as HIV and influenza [ 6 , 9 – 14 ]. For HIV, the development of bnAbs in infected individuals has been followed longitudinally for a number of donors [ 15 – 27 ]. It has become clear that bnAbs develop in natural infection as a result of the encounter of the humoral immune system with multiple different viruses corresponding to multiple different target Envelope (Env) molecules in a sequential fashion [ 13 , 28 , 29 ]. Typical Env molecules show little to no affinity for naïve precursor antibodies presented as receptors on the surface of B cells (BCRs) in the activation of bnAb lineages [ 12 , 30 – 37 ]. Usually, an approximation of the naïve B cell-triggering antibody is determined by computational methods extrapolating back from later time points in infection. The naïve antibody is often referred to as the unmutated common ancestor (UCA) of the bnAb lineage. Antibodies isolated during the course of infection are then analyzed with the goal of finding clues as to a set of immunogens that, when used sequentially, would elicit bnAbs [ 13 , 28 , 29 ].

When compared to other V3-glycan bnAb Fab structures, 11aa-insertion mAb HCs (17A, 23D and 50I) most closely resemble PGT135, which also has an insertion in CDRH1, while the LCs of these mAbs are more similar to that of PCDN-38B [ 23 ], with an RMSD of 0.48 Å ( S12(A) Fig ). The 4aa-insertion Fabs 50E and 55C could not be as readily compared to previously described N332-dependent bnAbs due to their CDRH3s, which are not completely resolved. Intriguingly, the 4aa-insertion Fab 50L differs substantially in structure from 11aa-insertion Fabs as well as from other V3-glycan Fabs due to a slightly more elongated conformation of its CDRH3 ( Fig 4 and S12(A) Fig ). A triad of Trp residues are located in the binding site of these antibodies; Trp residues are present in both of the CDRH1 insertions (11 and 4aa groups), but do not appear to be close enough to directly interact with the Trp residues found in either CDRH3 motif ( 100A WIG(E/D)L 100E or 100A WWAVE 100E ) ( S12(B) Fig ).

In contrast to the 11aa-insertion Fabs, structures of 4aa-insertion mAbs 50E and 55C revealed a more flexible and more difficult to resolve CDRH3 ( Fig 4D and 4E ). However, we were able to fully resolve the CDRH3 of another 4aa-insertion mAb 50L and show that CDRH1 participates in stacking interactions to stabilize the CDRH3 despite the shorter insertion length ( Fig 4F ). These differences in CDRH3 stability between the two antibody clades might explain contrasting neutralization potency despite similar breadth ( Fig 1C ).

(a) Ribbon representation of PC39-17A with 11aa CDRH1 insertion. HC CDRs are colored (CDRH1 in yellow, CDRH2 in orange, CDRH3 in red) and the 11aa insertion in CDRH1 shown in blue. Other regions of the HC are in lavender and the LC in grey in all panels. (b) Ribbon representation of PC39-23D Fab with 11aa CDRH1 insertion. (c) Ribbon representation of PC39-50I Fab with 11aa CDRH1 insertion. (d) Ribbon representation of PC39-50E Fab with 4aa CDRH1 insertion. (e) Ribbon representation of PC39-55C with 4aa CDRH1 insertion. (f) Ribbon representation of PC39-50L with 4aa CDRH1 insertion. (g) Ribbon representation of PC39-1 UCA with no insertion in CDRH1.

Structures of all three 11aa-insertion Fabs reveal an extended CDRH1 that provides support to and stabilizes the long 22aa CDRH3 (Kabat numbering; Fig 4A–4C and S12 Fig ) of the lineage. The similarity in backbone conformation (root-mean-square deviation (RMSD) = 0.3Å) and neutralization potency (0.10 vs. 0.09 respectively) of mAbs 17A and 50I suggests that the preconfigured interface in these structures is conserved across 33 months of Ab maturation. However, neutralization breadth was shown to increase during this time highlighting the importance of continued SHM ( S3 Fig ). The glycine at position G 100C found in the WIGEL motif is present in the UCA and only retained in 11aa-insertion Abs ( S2 Fig ). This sequence provides a glycine hinge to the unique mature bnAb CDRH3 structure.

Since the development of neutralization breadth was associated with an insertion of varying length at CDRH1, we sought to investigate the consequences of the insertion on antibody structure. X-ray crystal structures were solved for three 11aa-insertion Fabs (17A, 23D and 50I) and three 4 aa-insertion (group 4b) Fabs (50E, 55C and 50L) and the PC39-1 lineage UCA Fab. Each PC39-1 mAb is named after the timepoint (in mpi) at which it was isolated.

After viral escape, PC39-1 Abs continue to evolve to generate greater breadth of neutralization as assessed in the neutralization score out to at least 34 mpi ( Fig 2A–2C and S2 – S5 Figs). There is some decline in the neutralization score between 35 mpi and 55 mpi but this is small and may not be significant. One hypothesis to explain the observation of increased neutralization score after escape is continued affinity maturation in long-lived GCs [ 69 ].

Analysis of the prevalence of the deletion phenotype in the autologous viral population led to the observation that the Δ321 deletion variant is maintained in a single “arm” of the viral escape lineage but does not persist past 34 mpi in the other ( Fig 3E ). We looked for canonical V3-glycan resistance mutations in the lineage, and found that 324 GDIR 327 , N301 and N332 mutations occurred together in the other “arm” of virus evolution ( Fig 3E ). This pattern has been observed in other studies; 324 GDIR 327 residues are implicated in CCR5 co-receptor binding and the loss of the camouflaging glycans at N332 and N301 results in greater selection pressure on the GDIR motif [ 56 ].

Eventually virus escapes from bnAbs of the PC39-1 lineage and this occurs relatively precipitously at around 17 mpi ( Fig 3C ). To identify the mutations responsible for escape, we selected sensitive and resistant viral envelopes from time-points before and after escape ( S11(A)–S11(C) Fig ). Specifically, we included mutations that differed between our selected sensitive and resistant variants, as well as mutations occurring at any point in the autologous population that are known to affect V3-glycan Ab neutralization. Most strikingly, a deletion at 321 fully explained the difference in neutralization between the selected variants ( S11(A)–S11(C) Fig ). One hypothesis to explain the effect of this deletion is that it recesses the GDIR portion of the V3 loop, preventing contact with the antibody. Furthermore, reversion of the deletion in two early resistant viruses restores neutralization by PC39-1 Abs ( Fig 3D and S11(D) Fig ), confirming the importance of the deletion for neutralization escape.

Understanding in as much detail as possible the HIV Envs that triggered which bnAb lineages and how the triggering occurred could be valuable for HIV vaccine design [ 11 – 13 ]. The PC39-1 lineage results offer some potential insights. Early bnAb precursors are well defined in this case as are the predominant viruses around the time of the emergence of the lineage. Early bnAb precursors isolated via 10X that lacked insertions ( Fig 2 ) were synthesized, expressed and tested for binding by SPR to founder variant gp120 and NFL trimer proteins. No reactivity was found ( S10(A) and S10(B) Fig ). The bnAb precursors were then tested for neutralization of autologous viruses and showed no activity ( S10(C) Fig ). Similar findings were made for a computationally inferred PC39-1 UCA ( S10(D) Fig ). The data suggest that Env molecules in the case of infection and potentially clustered on a membrane may be able to trigger bnAb lineages at lower effective affinity for bnAb precursors than recombinant vaccine proteins, for which relatively high affinities may be required [ 68 ].

(a) Multidimensional scaling plot of PC39 virus sequences, colored by time point as shown. (b) Recombination plot representing individual env high-quality consensus-sequences (HQCSs) from PacBio sequencing as lines by time point, with each residue location colored by the most closely related founder variant as indicated. The top block shows amino acid positions that vary between the two founder variants in colors. (c) Matrix of IC 50 neutralization of autologous clones from PC39 at the given time point in mpi (rows) for PC39-1 mAbs (columns), colored as indicated. (d) IC 50 (top) and fold change (bottom) for PC39-1 Ab neutralization of the indicated autologous clones and the given mutations, colored according to the type of mutation. A three-letter code is used to designate residues from HXB2 positions 321 to 322 (HXB2 itself has a deletion phenotype, so has only 2 amino acids in the equivalent region), and a two-letter code in the mutant denotes a deletion at any position in the triad. (e) Multi-dimensional scaling plot of PC39 Env populations, colored as indicated by various motifs, with time mpi indicated by arrows.

Next, we sought to learn more about the virus(es) that had infected donor PC39 and their evolution through the course of infection. Viral sequencing and phylogenetic analysis suggested that two founder variants were involved in early infection. The variants differed by 6% under the GTR evolutionary model ( S9(A) Fig ). Due to founder variant recombination confounding further phylogenetic analysis, multidimensional scaling of viral sequences was employed to further investigate viral evolution. This analysis revealed a striking pattern of mixing between founders before subsequent independent evolution at around 17 mpi ( Fig 3A and 3B ). Investigation of sequences in the individual believed to have infected the PC39 donor revealed a diverse population of viruses that included related variants to the two founder variants ( S9(B) Fig ). We concluded that the epidemiologically linked yet substantially divergent founder variants are related, and that the source individual probably infected the donor with the two variants either at the same time or during a limited time-period.

Since the VH4-34 HC has been associated with self-reactivity [ 60 – 65 ] and since some of the mAbs isolated were arginine rich in the N-terminal region of the CDRH3, a feature associated with DNA reactivity [ 66 ], we investigated poly- and self-reactivity of a selection of PC39-1 lineage Abs. As shown in S8(A) and S8(B) Fig , with one or two exceptions, there was little evidence for any self- or polyreactivity. The UCA showed no reactivity with glycans on a NCFG microarray v5.5 [ 67 ] ( S8(C) Fig ), arguing that the lineage did not arise from an anti-glycan antibody, for example elicited against a glycosylated parasitic or bacterial antigen that then developed specificity for the protein part of its epitope on HIV Env.

The first attempt using the 10X platform yielded one HC-LC paired bnAb precursor (4.1% HC SHM and 4.6% KC SHM, no indels) at 9 mpi. We continued the methodology on the remaining time-points to isolate 52 mAbs of various CDRH1 insertion lengths ( Fig 2B–2D ). Monoclonal Abs isolated via 10X were found on the same phylogenetic branches as the mAbs isolated via flow-cytometry as well as new branches of the lineage that had only been previously sampled from bulk HC-only NGS ( S5(A) and S5(B) Fig ). A comparison of lineage frequency over time between bulk HC NGS and single-cell sequencing showed that the peak at 9 mpi in the NGS data was absent in the single cell data ( S5(D) Fig ). Gene expression analysis of 198,294 circulating memory B cells across 12 time-points identified three main B cell phenotypes (class-switched memory, atypical memory and antibody-secreting) that were defined based on preliminary annotation with CellTypist [ 57 ] as well as marker genes ( Fig 2C–2E and S6 and S7 Figs). This allowed us to speculate that the discrepancy between the lineage frequency based on bulk vs. single-cell methods was due to lineage plasmablast expansion at 9 mpi that was not present in the remaining time-points ( Fig 2C and 2D , S5(D) and S7(D) and S7(E) Figs). Plasmablasts with their high levels of antibody mRNA will greatly bias the bulk NGS lineage frequency but not that detected by single cell methods. Intriguingly, most lineage members were shown to have an atypical memory phenotype (42/52) followed by class-switched memory (8/52), and antibody-secreting (2/52). The atypical memory phenotype has been associated with chronic antigen exposure [ 58 , 59 ] and may suggest that continuous B cell engagement by Env contributed here to the development of breadth in the PC39-1 lineage.

The earliest Ab heavy chains from the lineage were detected at 9 mpi in the bulk HC NGS dataset, including a sequence with 99.4% identity to VH4-34*01/JH6*02 and lacking any CDRH1 insertion ( S2 Fig ). Reverting the single mutation in this HC, combined with a reverted germline LC sequence (iGL, see Methods ), gave us an unmutated common ancestor (UCA) ( S2 Fig ). This led us to hypothesize that the lineage began at or near 9 mpi due to the observed peak ( Fig 2A ) and we reasoned that it should be possible to isolate natively paired unmutated or near-germline members of the antibody lineage. However, our attempts to use flow-cytometry to isolate memory B cells at 9 mpi using the approach described above were unsuccessful and we turned to an alternate approach.

(a) The frequency of PC39-1 bnAb lineage transcripts identified from bulk heavy-chain NGS is shown longitudinally, as a percentage of total heavy chain transcripts (black line) with the contribution of each CDRH1 insertion length shaded in underneath and colored as indicated. Serum 37-virus panel neutralization score is shown as a dotted line. (b) Scatterplot of PC39-1 bnAb lineage mAbs isolated from both FACS and 10X HC (V H +J H ) vs LC (V K +J K ) somatic hypermutation (SHM, % nucleotide), colored by months-post infection of mAb isolation. The size of each dot indicates CDRH1 amino-acid insertion length. (c) UMAP projection of circulating memory B cell scRNA-seq clusters (198,294 cells; twelve time-points). (d) Tanglegram of corresponding HC (left) and LC (right) phylogenies for the PC39-1 bnAb lineage members isolated via 10X, colored by transcriptional phenotype as indicated. (e) Mean marker gene expression for B cell subsets. Dot size depicts the frequency of cells in which a gene is detected.

To investigate lineage maturation in more detail, we performed longitudinal bulk next-generation sequencing (NGS) of antibody heavy-chain transcripts from purified peripheral IgG+ memory B cells. The frequency of PC39-1 lineage transcripts over time revealed a large peak of expansion at 9 mpi followed by a rapid contraction concomitant with increased broadly neutralizing plasma activity ( Fig 2A ), a phenomenon that has been described for prior protocol C donors [ 21 , 23 , 24 , 27 ]. CDRH1 insertions could be detected as early as 11 mpi, and their frequencies in the PC39-1 lineage increased to completely dominate by month 55 with the two sublineages having a 4aa insertion being much more abundant than the sublineage with an 11aa insertion ( Fig 2A ). However, the presence of nearly every length of insertion from 1–11 amino acids ( S5(B) Fig shows 4, 5 and 11 aa insertions but insertions of length 1 (1 clones), 3 (5 clones), 6 (4 clones) and 8 (15 clones) aa were noted) could be found in the NGS HCs and were the result of adjacent sequence duplication. Somatic hypermutation within the lineage was found to increase over all time-points ( Fig 2B and S5(C) Fig ).

In common with many V3-glycan-directed bnAbs, PC39-1 mAbs were dependent on the N301-glycan, and specific interactions with residues of the 324 GDIR 327 motif at the base of V3, part of the co-receptor binding site, as well as a dependence on mutations in V1 ( S4(A) Fig ) [ 16 , 23 , 49 – 56 ]. Most mAbs were sensitive to D325 and R327 mutations, yet only those with the 11aa CDRH1 insertion were sensitive to mutation at every position in the 324 GDIR 327 motif ( S4(A) Fig ). This difference extended to gp120 binding, with 11aa insertion variants displaying a higher affinity for gp120s as measured by SPR and an absolute dependence on N332 ( S4(B) Fig ). Interestingly, several mAbs with a 4aa insertion and the single mAb with a 5aa insertion retained some binding affinity to N332 glycan-deleted gp120s as measured by ELISA, despite their dependence on N332 for neutralization.

The isolated mAbs alone did not fully recapitulate serum neutralization breadth, with the major difference being an inability to neutralize most CRF01_AE clade strains ( S1(C) Fig and S3 Fig ), likely attributable to the presence of a glycan at position 334 rather than 332 in this clade. Indeed, the only virus from this clade that was neutralized by PC39-1 mAbs retained a glycan at position 332 ( S3 Fig ).

A striking feature of the PC39-1 lineage is the occurrence, in all isolated mAbs, of an insertion in the HC CDR1 (CDRH1) at position 31 ( Fig 1C and S2 Fig ). The majority of mAbs contained either an 11 (residues 31A-K) or a 4aa (residues 31A-D) insertion paired with a 100A WIG(E/D)L 100E or 100A WWA(V/E)E 100E motif, respectively in the CDRH3, while only one isolated mAb had a 5aa insertion and a separate motif, 100A WVAVK 100E , in CDRH3. Two independent 4aa insertion events occurred within the developing lineage and can be found on two separate branches. The first 4aa insertion event (insertion 4a) resulted in a single branch predominantly defined by a ‘DYWD’ insertion in CDRH1 (there are two cases of a ‘TYWD’ insertion) and a 100A WWAEE 100E motif in CDRH3. The second 4aa insertion event (insertion 4b) resulted in two branches both overwhelmingly defined by a ‘SWND’ insertion (two cases of ‘AVND’ and one of ‘IWND’) in CDRH1 and a 100A WWAVE 100E motif in CDRH3.

To determine the predominant broadly neutralizing specificities of the sera, we investigated neutralization of select variants of viruses from different clades. A greatly reduced ability for donor plasma to neutralize N332A Env-mutated pseudoviruses of different clades identified the V3-glycan bnAb site as the major target for PC39 neutralization breadth ( Fig 1B ) [ 49 ]. To isolate monoclonal antibodies (mAbs) targeting the V3-glycan site, we affinity sorted B cells from PMBCs by flow cytometry selecting positively for reactivity with 92BR020 clade B gp120 and negatively with the corresponding gp120 N332A variant ( Fig 1B and S1(A) Fig ). The use of a clade B gp120 given that the donor was infected with a clade C virus strongly favored the selection of bnAbs. In total, 36 clonally related mAbs were isolated across eight timepoints displaying varying levels of neutralization breadth (43–76%) and potency (0.08–0.67 μg/mL IC 50 ) as measured on a 37-virus global panel ( Fig 1C and S1(B) and S1(C) Fig ). The bnAb lineage is defined by a CDRH3 length of 22 amino acids (aa), an IGHV4–34*01/IGHJ6*02 heavy chain (HC) and an IGKV3–20*01/IGKJ2*01 light chain (LC) gene ( Fig 1C and S2 Fig ). We termed this lineage PC39-1.

(a) Longitudinal development (months) of heterologous serum neutralization. Serum neutralization breadth, defined as the % of viruses showing >50% inhibition of infectivity at the lowest plasma dilution of 1:50 using a 37-virus indicator panel, is shown in pink; neutralization score is shown as a black line and viral load as a red line. (b) Neutralization Inhibitory Dilution 50 (ID 50 ) for a heterologous isolate, 92BR020, and an N332 knockout variant are shown in black and green, respectively, for each time point. (c) Characteristics of PC39-1 mAbs include insertion length relative to unmutated ancestor sequence, HC complementarity determining region 1 (CDRH1) insertion sequence, CDRH3 and CDRL3 sequences, HC and LC V- and J-gene nucleotide somatic mutation percentage, (%SHM), neutralization breadth and potency on two panels (colored as indicated; breadth is defined as viruses with IC 50 < 50 μg/mL in TZM-bl pseudovirus assay, potency is defined as the geomean IC 50 in μg/mL of neutralized viruses). Abs are organized and colored by clade identity based on CDRH1 insertion and named based on the time point (in mpi) from which they were isolated. The HC phylogeny for the PC39-1 mAbs is shown on the left and colored by antibody clade.

IAVI Protocol C is a large cohort of individuals in sub-Saharan Africa identified during primary HIV infection and then followed longitudinally, examining sera for the development of neutralization breadth [ 47 ]. Donor 39 (PC39), infected with a clade C virus and followed for more than 5 years, showed exceptional cross-clade neutralization breadth when tested against a global panel of 37 viruses ( Fig 1A ). The development of measurable breadth became clear at around 9–11 months post infection (mpi), expanded rapidly between 11 and 20 mpi and levelled off at >80% around 23 mpi. The potency of serum neutralization as reflected in a neutralization score [ 48 ] continued to increase out to around 34 months before levelling off ( Fig 1A ).

Discussion

We describe here an HIV infected individual who developed serum antibody neutralization breadth incorporating several interesting features that may shed light on strategies for bnAb-targeted vaccine design.

The infection course of donor PC39 was unusual in that two distinct but related founder viruses were established early in the individual. The two viruses either infected the individual in a single transmission event or from repeated exposure to the infecting partner early in the infection. The related founder viruses then underwent extensive recombination, and this occurred concomitant with the initiation and development of neutralization breadth. The development of breadth under these circumstances is consistent with observations of a higher rate of neutralization breadth in superinfection in some cases although not others [18, 25, 38–42] and of the association of neutralization breadth with multiple founder viruses [43]. The latter is particularly comparable to the current case and lends some support to the observations of the authors of that previous study and their suggestion for an HIV vaccine strategy: “These results demonstrate that the presence of slightly different HIV-1 variants in acute infection could promote the induction of bnAbs, suggesting a novel vaccine strategy, whereby an initial immunization with a cocktail of minimally distant antigens would be able to initiate bnAb development towards breadth”. Having undergone extensive recombination, two virus arms then emerged at around 17 months and these two arms showed distinct features including the details of neutralization escape.

In our longitudinal study, neutralization escape occurred rapidly once the PC39-1 bnAb lineage emerged, but the lineage continued to develop in terms of neutralization potency for several months. This is consistent with ongoing affinity maturation in long-lived germinal centers (GCs) as has been described [69]. It is tempting to speculate that the sequestration of multiple related viruses on FDCs in GCs might favor the maturation of bnAbs, for example by enhancing internalization of antigen that could be achieved relative to nAbs with narrower specificities.

The initiation of neutralization breadth coincided with the appearance of indels in the CDRH1 of PC39-1 lineage Abs. A series of independent insertion events occurred at the same position in CDRH1 to yield clones with insertions of lengths varying from 1–11 amino acids. The predominant insertion lengths were 4 and 11 amino acids with the former arising from two independent events and showing different characteristics. The structural importance of the 11 amino acid CDRH1 insert appears to be to stabilize the long CDRH3 of the PC39-1 lineage Abs. For the 4 amino acid CDRH1 insert, stabilization is less apparent. However, in both 4 and 11 amino acid cases, the acquisition of a triad of tryptophan residues from CDRH1 and CDRH3 is observed, but it is not yet clear what the functional and evolutionary consequences are of this particular feature. HIV immunogen design based on insights gained from bnAbs (reverse vaccinology 2.0 [3, 8]) has tended to prefer bnAbs without insertions or deletions (indels) with the understanding that this requirement may increase the difficulty of inducing bnAbs in a sequential immunization strategy [13]. The frequency of insertions in the IgM memory and IgG memory of healthy individuals is of the order of 2% [44–46] with a decreasing frequency as the insertion length increases (S13 Fig). The demonstration here of multiple independent insertions at a sensitive site suggests that there may be opportunities for the generation of such insertions if an appropriate antigen stimulation via vaccination can be achieved. Given that V3-glycan bnAbs interact with and/or accommodate glycans, there was a formal possibility that the PC39-1 lineage arose from an initial clone generated against a glycan moiety. Our study showed no evidence for glycan reactivity in lineage precursors. Another concern that arose was that the PC39-1 VH gene segment used is VH4-34, which has been associated with self-reactivity. However, there was little evidence for self-reactivity of the great majority of PC39-1 lineage members. Therefore, it seems most probable that the lineage was triggered by the Env antigen itself.

Overall, this study, together with another recent study [43], suggests a potential advantage for inducing bnAbs in an immunization strategy including multiple related Env (MRE) molecules. Of note, the studies describe the development of breadth after exposure to multiple founder viruses, and their recombinants in our case, that are closely related but not as similar generally as the swarm of viruses predominating at any one-time during cases of acute infection with a single virus. Indeed, Keele et al. concluded, from a cohort of 102 subjects with acute HIV infection, that individuals infected with a single virus showed env diversity ranging from 0.08 to 0.47% while individuals infected with multiple related viral variants from a single source partner (either in the same transmission event or multiple transmission events over time) showed env diversity ranging from 0.86 to 6.63% [70]. Therefore the env diversity of PC39 at 6% at early time-points is in the upper range of previously reported numbers for multiple related variant infections. Interestingly, Piantadosi et. al found a positive correlation (Spearman’s rho = 0.51, P = 0.008) between env diversity at acute infection and nAb breadth at 5 years post-infection in a cohort of 26 non-superinfected individuals who had a median env breadth of 0.28% (range, 0 to 4.0%) [71]. However no association was found between gag diversity at acute infection and nAb breadth at 5 years post-infection. This study again supports a possible role for early encounter with MRE in the development of neutralization breadth.

It should be noted however that, in order to make the claim that increased serum nAb breadth in donors infected with multiple related viruses is due to true bnAb development and not a polyclonal response consisting of multiple isolate specific nAbs, targeting of bnAb epitopes must be shown. Futhermore, the nAb response elicited by different founder viruses should be shown to be the same. Both demonstrations were made above for PC39 bnAbs. Still, questions remain—is PC39 bnAb development due to a prime-boost in which the individual was infected with two variants over multiple time-points or is it due to a cocktail effect in which the bnAbs develop against MRE molecules in a single germinal center? Or both? The development of bnAb breadth as the result of BCRs reacting with a cocktail of recombinant viruses seen between 9 and 17mpi is consistent with initiation of the lineage at 9mpi and viral escape at 17mpi.

A potential limitation of our study is the lack of available sequences from acute infection. As a consequence, we are unable to clearly define the true founder viruses from donor PC39. However, although advanced techniques for inferring founder multiplicity exist [72], our study has convincingly demonstrated that the initial variants sequenced from donor PC39 exhibit a close genetic relationship to different, distinct variants identified within the diverse source donor population (as illustrated in S9(B) Fig). Therefore, despite this caveat, our findings strongly support the idea that the PC39-1 bnAb lineage is not a consequence of superinfection, but rather the outcome of interactions with MRE from related founders.

In contrast to infection with multiple closely related founder viruses, superinfection typically shows greater sequence differences between infecting viruses and has not been clearly associated with the development of neutralization breadth [18, 25, 38–42]. Within a given host, HIV env diversity in cases of superinfection can range from 10% (for inter-subtype superinfection) to >30% (for intra-subtype superinfection) [73]. Therefore, there is a suggestion that there may be a “sweet spot” in terms of the relatedness of immunogens in a cocktail to favor the development of breadth. Indeed, modeling studies [74–76] have focused on the importance of mutational distance between antigens in a putative cocktail. The authors have suggested that, when variant antigens are separated by only small mutational differences, then strain specific antibodies develop. When variant antigens are too different then affinity maturation cannot be sustained and few antibodies result.

Finally, in terms of activating appropriate bnAb precursors, there is a concern about the frequency of such precursors in the naïve B cell repertoire so that immunization will be effective in the overwhelming majority of vaccinees [77–79]. This concern may not be well addressed by the MRE strategy. However, if appropriate germline activating immunogens are designed [30, 34–36] then the MRE strategy may be worthy of investigation as a boosting step in a sequential immunization protocol.

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[1] Url: https://journals.plos.org/plospathogens/article?id=10.1371/journal.ppat.1011416

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