Membrane-bound mRNA immunogens lower the threshold to activate HIV Env V2 apex-directed broadly neutralizing B cell precursors in humanized mice

Summary Eliciting broadly neutralizing antibodies (bnAbs) is the core of HIV vaccine design. bnAbs specific to the V2-apex region of the HIV envelope acquire breadth and potency with modest somatic hypermutation, making them attractive vaccination targets. To evaluate Apex germline-targeting (ApexGT) vaccine candidates, we engineered knockin (KI) mouse models expressing the germline B cell receptor (BCR) of the bnAb PCT64. We found that high affinity of the ApexGT immunogen for PCT64-germline BCRs was necessary to specifically activate KI B cells at human physiological frequencies, recruit them to germinal centers, and select for mature bnAb mutations. Relative to protein, mRNA-encoded membrane-bound ApexGT immunization significantly increased activation and recruitment of PCT64 precursors to germinal centers and lowered their affinity threshold. We have thus developed additional models for HIV vaccine research, validated ApexGT immunogens for priming V2-apex bnAb precursors, and identified mRNA-LNP as a suitable approach to substantially improve the B cell response.


In brief
Eliciting broadly neutralizing antibodies (bnAbs) to HIV, such as the V2-Apextargeted bnAb PCT64, is the goal of germline-targeting (GT) vaccines. Using humanized Ig knockin mouse models, Melzi et al. demonstrate the activation of rare PCT64 precursors with a high-affinity immunogen, ApexGT5. Furthermore, they find that mRNA-LNP-encoding membrane-bound ApexGT5 trimers lowers the affinity threshold for activation relative to protein immunogens.

INTRODUCTION
Despite decades of research, an HIV vaccine remains elusive (Ng'uni et al., 2020), but the discovery that some HIV-infected individuals can develop broadly neutralizing antibodies (bnAbs) capable of potently neutralizing a high proportion of HIV-1 isolates has revolutionized the field (Klein et al., 2013;Flemming, 2018;Sok and Burton, 2018). These bnAbs target highly conserved regions of HIV envelope glycoprotein (Env), including the V2-apex region (Burton and Hangartner, 2016;Sok and Burton, 2018).
Previously, we reported an approach to design GT immunogens to bind precursors of HCDR3-dominant bnAbs, which triggered robust responses from BG18 precursors (Steichen et al., 2019). V2-apex directed antibodies, such as PG9, PG16, CAP256, and PCT64, are also heavily reliant on HCDR3 for neutralization and thus ideal targets to determine whether this strategy is generalizable to another Env epitope. Most V2-apex bnAbs have long (R25 residues), protruding, anionic, and often tyrosine-sulphated HCDR3 loops to penetrate the glycan shield and reach a positively charged glycopeptide epitope on the Apex of Env (Walker et al., 2009;Pancera et al., 2013;Doria-Rose et al., 2014;Andrabi et al., 2015). Notably, V2-apex bnAbs are among the most commonly identified bnAb families in patient serum samples, arise early post-infection Georgiev et al., 2013;Landais et al., 2016), and require only moderate SHM, making them highly desirable targets for immunization . Of particular interest is PCT64; isolated from an HIV-1 subtype A-infected donor (Landais et al., 2016(Landais et al., , 2017, PCT64 neutralizes 29% of all HIV isolates with moderate potency and up to 56% and 48% of subtype A and C viruses, respectively (Landais et al., 2017). It has an HCDR3 length of 25 amino acids and 10%-12% SHM; compared to other V2-Apex bnAbs, its HC precursors are relatively common in the human repertoire (upper limit of $20 in 1 3 10 6 B cells) (Willis et al., 2022). Furthermore, the maturation trajectory of the PCT64 antibody line has been described in a three-year-long coevolutionary study that offers a blueprint for recapitulation (Haynes et al., 2016;Landais et al., 2017;Rantalainen et al., 2018).
Here, we developed two preclinical mouse models with B cells expressing two distinct early ancestors of PCT64 to assess protein-and mRNA-based Apex germline-targeting (ApexGT) immunogens (Willis et al., 2022). High affinity ApexGT immunogens activated rare PCT64 precursors and induced on-track mature PCT64-like mutations, with evolution driven primarily by the HC. Furthermore, we found that mRNA-LNP mediated in vivo expression of a membrane-anchored ApexGT trimer immunogen was a promising avenue for priming PCT64-like responses.

Generation of a PCT64 precursor knockin mouse
To study immune responses against the Apex of HIV-1 Env in vivo, we generated a knockin (KI) mouse with B cells bearing the least mutated common ancestor (LMCA) heavy chain (IGH) and light chain (IGK) of bnAb PCT64, which was isolated from a human donor (Landais et al., 2017). PCT64 LMCA IGH has 99.4% germline sequence identity with a fully reverted germline V gene (VH3-15*01) and a J gene (JH6*03) containing three amino acid mutations, while the PCT64 LMCA IGK is encoded by a fully germline V gene (Vk3-20*01) and J gene (Jk3*01) (Landais et al., 2017).
Using our CRISPR/Cas9 protocols Wang et al., 2021), we inserted the PCT64 LMCA IGH and IGK variable regions into their respective native murine loci and confirmed insertion by genotyping. Next, we established the frequency of cells expressing the PCT64 KI sequences by sorting and sequencing B220 + naive peripheral B cells ( Figure S1A). PCT64 LMCA IGH was expressed by 85.7% of naive B cells (Figure S1B) and PCT64 LMCA IGK by 86.3% ( Figure S1C). Paired sequences showed that both human PCT64 LMCA IGH and IGK could form hybrid BCRs by pairing with a variety of murine heavy chains (HCs) and light chains (LCs) (Figures S1D and S1E). To generate mice carrying the full PCT64 LMCA antibody, we crossed PCT64 IGH (PCT64 LMCAÀH ) and PCT64 IGK (PCT64 LMCAÀK ) mice and obtained offspring where both human IGH (96%-100% of expression) and IGK (86%-90%) paired with each other ($81%) (Figures 1A, S1F, and S1G). Screening differentiation stages in the bone marrow (Figures S2A and S2B) and in the spleen (Figures S2C and S2D) confirmed that B cells underwent normal development in both the PCT64 LMCAÀH and PCT64 LMCA mice (Hardy et al., 1991). To assess whether PCT64 LMCA B cells express functional BCRs, we measured antigen specific binding to an ApexGT2 trimer probe (GT2 below) engineered to bind PCT64 LMCA with moderate affinity (monovalent K D = 167 nM) (Willis et al., 2022). Approximately 58% of peripheral blood B cells bound to GT2 in a double fluorophore staining assay ($0.04% of B cells in C57BL/6J mice were GT2 reactive); 99% of binders were found to be epitope-specific using an epitope knockout (KO) probe (GT2-KO) ( Figures 1B and 1C). B cell receptor (BCR) sequencing found GT2-reactive B cells to be positive for both the human PCT64 LMCA IGH (100%) and corresponding human PCT64 LMCA IGK (95%) ( Figure 1D). In summary, PCT64 LMCA B cells exhibited Ig gene allelic exclusion, underwent normal development, and expressed functional paired human KI BCRs, which bound the ApexGT2 probe.   Figure 1E) to decrease precursor frequency and facilitate the tracking of PCT64 LMCA responses. We immunized mice 24 h after adoptive transfer with either GT2 or BG505-MD39 SO-SIP trimers formulated in Sigma adjuvant system (Sigma) intraperitoneally (i.p.)-an established route for SOSIP immunogens Steichen et al., 2016)-and measured responses in the spleen ( Figures 1E-1G and S2E). GC responses (GC; CD95 hi CD38 lo ) were detected 8 days post immunization (dpi) in all groups; CD45.2 + PCT64 LMCA B cells represented on average 3.7% of the activated GC B cells in GT2 immunized mice at this timepoint, and 63% were GT2 specific ( Figure 1F). CD45.2 + cells were not detected in GCs in the BG505-MD39 SO-SIP trimer cohort or in mice that received WT cells ( Figure S2E). At 16 and 42 dpi, CD45.2 + GC responses were still ongoing. The GT2 specificity of CD45.2 + GC cells increased from 8 to 42 dpi (from 62.8% to 79.4%, p = 0.0038), suggesting ongoing selection expanding clones with higher affinity for GT2 ( Figure 1G). Furthermore, epitope-specific IgG titers were detectable at 7 dpi in PCT64 LMCA recipient mice and gradually declined after a peak at 14 dpi ( Figure 1H). WT mice immunized with GT2 trimers failed to develop epitope-specific IgG antibodies. No off-target antibody responses against epitope-deficient GT2 trimers (GT2-KO) were detected in either group ( Figure 1H), indicating that serum IgG antibody responses were due to the transferred PCT64 LMCA B cells.
Overall, PCT64 precursors were successfully activated by GT2 immunization, formed sustained GC reactions, and generated epitope-specific IgG responses.
GT2-primed PCT64 LMCA BCR heavy chains acquire bnAb-like mutations To determine whether PCT64 LMCA B cells underwent SHM and accumulated PCT64-like mutations after GT2-trimer immunization, we sorted class-switched GT2 + PCT64 LMCA B cells at 8 and 42 dpi for single-cell BCR sequencing. Tracing lineage evolution of the PCT64 LMCA IGH highlighted broad diversification at 42 dpi ( Figure 2A). SHM was minimal in the PCT64 LMCA IGH and IGK V-region at 8 dpi but increased over time ( Figure 2B). At 42 dpi, there was significantly higher SHM in the IGH V region (8.6 nucleotides [nt]/5.6 amino acids [aa]) than in the corresponding IGK V region (5.9 nt/4.5 aa) whether comparing nt (p = 0.0013) or aa (p = 0.037) ( Figure 2B). Mutations accumulated over time in recurring positions in the HCDR1, HCDR2, and HCDR3 (Figure 2C). Substitutions at these sites were enriched for aa present in the mature PCT64 bnAb (V-region position 31, 35, and 52B) and for aa present in early PCT64-line isolates (in HCDR3 position 100D) ( Figure 2D) (Landais et al., 2017). N31D, which is also present in the mature PCT64 bnAb (Landais et al., 2017), was acquired in 98% of the isolated HC sequences, indicating positive selection. While enriched mutations in the HC V region were likely facilitated by activation-induced deaminase (AID) binding sites, no underlying AID hotspot was identified for the HCDR3 mutation (100D) ( Figure S2F).
In contrast, no mutations were enriched in the PCT64 LMCA IGK ( Figure 2E), suggesting an HC-driven immunogen interaction. To confirm this SHM drove antibody maturation, we expressed 14 representative 42 dpi Fabs and quantified their affinity for GT2. Of the 10 Fabs with detectable affinity for GT2, 9 showed increased affinity over the PCT64 LMCA Fab (K D , 130 nM). The geomean K D for all 10 Fabs (4.3 nM) indicated a 30-fold improvement; one Fab exhibited a 394-fold affinity gain over the native PCT64 LMCA Fab. ( Figure 2F).
To interrogate the interaction between the acquired mutations and GT2, we determined the cryo-EM structure of GT2 in complex with a high affinity day 42 Fab (GT2-d42.16) (Figures S3A-S3C). The Env base-binding Fab RM20A3 was included to improve particle angular distribution. Our $3.5 Å -resolution reconstruction ( Figures S3D-S3G) allowed us to build atomic models of the complex ( Figure 2G), which confirmed that GT2-d42.16 adopts a nearly identical structure and angle of approach to PCT64 LMCA ( Figure 2H) with the characteristic elongated and anionic HCDR3 beta hairpin loop that extends inward toward the 3-fold axis of the trimer apex and extensive engagement with positively charged residues in the V1/V2 loop and the glycan at N160gp120 A ( Figures 2G, S3H, and S3I). Although the map resolution is lower due to inherent flexibility in the distal regions of the Fab outside of the paratope ( Figure S3C), docking of the high-resolution PCT64 LMCA crystal structure allowed us to reliably identify the sites of SHM acquired during affinity maturation ( Figure 2H). The only positions of SHM within the paratope are in the HCDR1 and 2 domains, which are responsible for engaging/ accommodating the glycan at N156gp120 A as well as the C strand of V2 ( Figure 2I) (Willis et al., 2022). Although the N156gp120 A glycan does not engage in any specific sidechain contacts, it forms several backbone hydrogen-bonds (H-bonds) ( Figure 2I) that could be enhanced by the HCDR1 and/or HCDR2 mutations by stabilizing the small helical turn in the HCDR2. Both the K52 b N and T52 c I mutations are present in early PCT64 lineage members such as PCT64.13B, although both sites were further mutated in the mature PCT64 bnAbs (Landais et al., 2017). The N31D mutation, located near the interface with the C strand, is present in the majority of mature PCT64 lineage members and could contribute to a more favorable electrostatic interaction with the positively charged apex. In sum, GT2 immunization could successfully initiate PCT64 LMCA maturation toward a higher affinity and mature-PCT64-like antibody.
PCT64 precursor responses to GT2 are driven by the heavy chain In Apex-directed bnAbs, the HCDR3 is a major binding determinant (Pancera et al., 2010;Pejchal et al., 2010;McLellan et al., 2011;Julien et al., 2013;Andrabi et al., 2015). Mammalian display directed evolution was used to engineer the GT2 trimer to target the PCT64 LMCA HC, similar to the strategy used for targeting N332-dependent bnAbs (Steichen et al., 2019). To validate the specificity of the GT2 immunogen for PCT64 IGH, we used a PCT64 LMCA HC-only mouse model (PCT64 LMCAÀH ) in which the human PCT64 LMCA IGH pairs with native murine LCs.
Next, we quantified antigen-specific binding to GT2 trimer probes in naive PCT64 LMCAÀH mice. Approximately 32% of peripheral blood B cells bound the GT2 probe in a double fluorophore staining assay ( Figure 3B); binders consisted of 100% human PCT64 LMCA IGH paired with various murine LCs ( Figure 3C); hybrid BCRs were thus capable of GT2 binding.
Due to this enrichment, we measured the affinities of PCT64 LMCA IGH paired with different naive murine IGK (Figure 3F). Hybrid antibodies isolated from naive PCT64 LMCAÀH mice, which constituted a polyclonal population generated through LC variability, had affinities (K D s) from 1.6x10 À6 to 2.5x10 À8 M. Some IGK V genes, such as IGKV2-109 and IGKV12-44 (both enriched in the GC), had up to 2.6-fold higher affinity for GT2 than PCT64 LMCA paired with its natural human LC. In contrast, lower affinity was found with IGKV1-135 and IGKV2-137, which were far less frequent in the GC than in the naive repertoire ( Figure 3F).
We then investigated whether IGH could acquire PCT64-like mutations in the absence of the human LC. SHM and diversification increased over time ( Figure S5B); by 8 dpi, human VH3-15 acquired an average of n = 1.2 ± 1.6 nt mutations (0.89 ± 1.1 aa) ( Figure S5C) and reached an average of n = 7.7 ± 2.4 nt (4.9 ± 1.9 aa) by day 42 (Figure S5C), similar to the rate observed in the full PCT64 LMCA KI model. Enriched residues in PCT64 LMCAÀH IGH matched those identified for PCT64 LMCA (Figures 3G, 3H, S5D, and S5E), and the aa mutations at these sites included previously identified PCT64-like mutations in positions 31, 35, 52 B , and 100 D ( Figures 3G and 3H), suggesting that IGH evolution in response to GT2 trimers is both consistent and independent of the LC. This demonstrated that B cells bearing PCT64 LMCA HC in conjunction with diverse LCs could respond with great specificity to GT2 immunization.
A high affinity immunogen is required to activate rare PCT64 precursors Human repertoire data suggest that a suitable PCT64-immunogen needs to reproducibly trigger B cells at frequencies lower than $20 precursors per 10 6 (Willis et al., 2022). To evaluate the capacity of GT2 to activate PCT64 precursors at human physiological frequencies, we calculated the frequencies of GT2 + PCT64 LMCAÀH B cells in the spleens of recipient mice at the time of immunization, 24 h after the adoptive transfer of 5x10 5 , 1x10 5 , or 5x10 4 CD45.2 + B cells ( Figures 4A and 4B). The resulting GT2-specific CD45.2 + B cell frequencies were 100, 20, or 10 per 10 6 splenic B cells, respectively ( Figures 4C and 4D). Responses in immunized recipient mice with defined numbers of PCT64 LMCA B cells were analyzed 8 dpi ( Figure 4E). While PCT64 LMCA frequency did not affect total GC size, it did affect the proportion of CD45.2 + cells in GCs, from 1.2% CD45.2 + at 100:10 6 to barely 0.2% at 10:10 6 ( Figures 4F and 4G).
Immunogen affinity is key to rare B cell activation (Dosenovic et al., 2015;Sok et al., 2016;Tian et al., 2016;Abbott et al., 2018). To assess the effect of affinity on activation, we immunized mice with the range of PCT64 precursors defined above with ApexGT5 (GT5 below), an ApexGT trimer with higher affinity for PCT64 LMCA (K D , 66 nM compared to 167 nM for GT2) (Figure 4H) (Willis et al., 2022), and compared responses at 8 dpi to GT2 ( Figure 4E). We observed a relative increase in CD45.2 + B cell recruitment to GCs in all GT5-immunized groups. The gap was most pronounced at the lowest precursor frequency (10:10 6 ) in which GT5 immunization activated 20 times more PCT64 LMCA B cells than GT2 (4% CD45.2 versus 0.2%) ( Figures 4I and 4J). GT5-specific responses were dominated by CD45.2 + B cells (>90%) at all tested precursor frequencies; in contrast, GT2-specific responses were directly proportional to the initial precursor number and were outnumbered in the GC by competitor CD45.1 + murine B cells (Figures S5F and S5G).
To assess GT5 efficacy at even more stringent frequencies, we established cohorts with 100, 10, or 1 precursor(s) per 10 6 . While GC responses decreased over time, CD45.2 + cells persisted in the GC from 8 until 42 dpi at both 100 and 10 per 10 6 ( Figures 4K and 4L). However, at 1 per 10 6 , only a weak CD45.2 + response was generated at 8 dpi, and none was    Article detected by 42 dpi. Epitope-specific IgG responses were detected by ELISA from 7 to 42 dpi in recipients of 10 PCT64 LMCA B cells per 10 6 ; GT2-immunization produced comparable titers in recipients of 100 PCT64 LMCA B cells per 10 6 . WT control mice immunized with GT5 did not develop detectable epitope-specific IgG responses, and no off-target response was detected in any group ( Figure 4M). Thus, the higher affinity GT5 protein trimer could specifically activate PCT64 precursors at frequencies approaching the estimated human physiological range.
GT5 immunization induces mature PCT64-like mutations in IGH To determine whether GT5 could induce on-track SHM in PCT64 precursors, we sorted CD45.2 + GT5 + IgG1 + B cells at 42 dpi for single-cell BCR sequencing ( Figure S5H). Diversification occurred in both PCT64 LMCA IGH and IGK sequences ( Figure 5A); the average number of mutations acquired in the V-region was 6.6 nt/4.4 aa for IGH and 6.2 nt/4.6 aa for IGK ( Figure 5B). From this antibody library, we expressed 15 representative Fabs; 13 of 15 (87%) showed detectable binding to GT5 (K D <10 mM), with a geomean affinity of 0.59 nM among binders, representing an $110-fold increase over the affinity of the PCT64 LMCA (66 nM). We also produced 13 Fabs with PCT64 LMCA IGH paired with murine IGK chains; 9 of 13 (69%) had detectable affinity, and the geomean affinity among binders was 0.29 nM, an even larger increase over PCT64 LMCA ( Figure 5C). We compared mutations acquired by IGH in our model with those from human PCT64 precursors isolated 8-35 months post-infection to determine whether the trajectory of antibody evolution was similar (Landais et al., 2017). IGH sequences isolated at 42 dpi carried an average of 3-4 PCT64-like mutations and a peak of 7 PCT64-like mutations ( Figure 5D). Total mutations and PCT64-donor-like mutations were positively correlated (R 2 = 0.4869). GT5 immunization generated a broader, more diverse repertoire than GT2; aa mutations in IGH were distributed across more numerous sites, though relatively less enriched ( Figures 5E and 5F). This plasticity promoted PCT64-like mutations at 3 sites in HCDR1 (positions 28, 31, and 35), 3 sites in HCDR2 (positions 52, 52B, and 52C), and 4 sites in HCDR3 (positions 92, 97, 100C, and 100D) ( Figure 5F). As with GT2, no enrichment site was identified in the LC.
To assess whether SHM acquired from priming conferred a degree of neutralization, five GT2-and five GT5-induced day 42 mAbs were tested against a series of WT and modified (with ApexGT mutations) HIV pseudoviruses (PSVs) based on isolates neutralized by PCT64 ( Figure 5G) (Landais et al., 2017). None of the day 42 mAbs could neutralize the WT PSVs, but some showed partial neutralization of PSVs with ApexGT mutations, especially PSVs with the K169R mutation (PSVs GT5-V2B and GT5-N167) ( Figure 5G). This is consistent with the binding mode of PCT64 LMCA (Willis et al., 2022) and day 42 Fabs (Figures 2G-2K and 5H-5L), which includes strong electrostatic interactions between their acidic residues and R169 of ApexGTs. To clarify the molecular details of GT5-induced SHM, we determined the cryo-EM structure of GT5 in complex with a high-affinity day 42 Fab (GT5-d42.16) ( Figures 5H and S6A-S6G). Despite positional differences in the location of SHM sites (magenta), no structural differences were detected between GT5-d42.16 and either PCT64 LMCA or GT2-d42.16 ( Figures 5H and S6H-S6M). GT5-d42.16 has five more sites of SHM than GT2-d42.16 while both Fabs present SHM in the HCDR2 domain, which is responsible for engaging/accommodating the N156gp120A glycan and the C strand of V2 ( Figures 5H and 5I). The glycan at N156 engages in multiple backbone H-bonds with GT5-d42.16, while E53 forms an H-bond with GT5 residue K171, as in GT2-d42.16. Notably, the D53E mutation is present in the vast majority of PCT64 lineage members. Unlike GT2-d42.16, GT5-d42.16 has one site of SHM in the HCDR3 domain, T93 N, which is located at the very beginning of the loop near the N160gp120 A glycan binding pocket and is also found in the PCT64.35M bnAb lineage. All remaining mutations in the HC are located outside the paratope and thus not directly involved in affinity maturation. GT5-d42.16 also has several LC mutations, with one, S31I, located near the N160gp120 A glycan binding interface in the LCDR1 domain ( Figure 5H). However, this residue converges on either an Asn or Asp in all the mature PCT64 antibodies.
At the immunogen level, the only difference between GT2 and GT5 is the identity of loop2B ( Figure 5H). The GT5 loop2B has the N187 glycan knocked out and includes a mutation to a bulky tryptophan residue at position 188 at the tip of the loop, among other mutations. Using difference mapping, we found that the removal of the N187 glycan creates a hole in the density Article surrounding the PCT64 binding site that results in a slight change in the average binding angle of the Fab, presumably by relieving steric restrictions from the glycan ( Figure 5J), which is in line with the surface plasmon resonance (SPR) results showing a slightly faster on and off rate of PCT64 LMCA for GT5 (Willis et al., 2022). The conformation of the loop is also affected, especially on protomer C, where it is folded inward toward the Fab and N160gp120 C glycan ( Figures 5K and 5L). Although W188 does not appear to interact with the GT5-d42.16 HCDR3, there is clear electron microscopy (EM) map density extending from the tip of the loop to the N160 glycan resulting from the W188 residue, suggesting it could be stabilizing the glycan, and, in turn, its interactions with the Fab ( Figure 5K). Thus, immunization with GT5 can induce affinity maturation in rare PCT64 precursors and support the acquisition of PCT64-like mutations that neutralize autologous virus.

mRNA-LNP membrane-bound GT5 trimers potently activate PCT64 precursors
Nucleoside-modified mRNA vaccines for SARS-CoV-2 have proven safe and highly effective in humans (Baden et al., 2021;Thomas et al., 2021). Thus, GT5 was further developed as a membrane-bound trimer with appropriate antigenic profile expressed from DNA or mRNA (Willis et al., 2022). As human vaccines are frequently administered intramuscularly (IM) (Zhang et al., 2015), we first assessed IM GT5 protein trimer delivery. We established PCT64 LMCA at 10 per 10 6 B cells in recipient mice, immunized IM with GT5 in Sigma adjuvant, and measured the response in the inguinal lymph nodes (LN) at 13, 28, and 42 dpi ( Figure 6A). GT5-specific CD45.2 + B cells were undetectable in 13 dpi GCs and very infrequent at later timepoints ( Figures 6B  and 6C). To assess whether B cell responses could have occurred at other sites, we quantified GT5-specific IgG serum by ELISA but retrieved no detectable titers ( Figure 6D). GT5 protein trimers may, therefore, not be suited for IM delivery in mice. We then evaluated immune responses to mRNA-mediated expression of membrane-bound GT5 trimers. Recipient mice (10:10 6 ), which were immunized IM with a single dose of mRNA-LNP encoding GT5, had large GCs and high recruitment of CD45.2 PCT64 LMCA B cells at 13 dpi ( Figures 6E and 6F). PCT64 LMCA B cells were maintained in GCs up to 28 and 42 dpi, and GT5-specificity increased from 60% to 90% of CD45.2 GC B cells over time (Figures 6E and 6F). ELISA of serum IgG titers found that IM mRNA-LNP immunization induced longlasting, GT5-specific antibodies ( Figure 6G).
Given the magnitude of the response to mRNA immunization, we tested whether GT5-mRNA could generate consistent responses after a 10-fold precursor reduction to 1 per 10 6 B cells (Figures 6H and 6I). Even at this extremely rare starting frequency, PCT64 LMCA averaged 4% of GC B cells at 13 dpi (in comparison to 9.9% at 10:10 6 ) and expanded to 26.6% at 42 dpi ( Figure 6I). To confirm that mRNA-LNP GT5 maintained the capacity to induce PCT64-like mutations, we performed BCR sequencing of class switched CD45.2 + GT5 + B cells at 42 dpi. Isolated IGHV genes acquired an average of 7 nt/5 aa mutations ( Figure 6J). Enriched sites occurred in similar positions previously identified with protein trimer immunization ( Figure 6K).
Thus, mRNA-LNP-encoded GT5 trimers activated ultra-rare PCT64 precursors more potently than soluble protein trimers and induced the acquisition of similar bnAb-like mutations.
mRNA-LNP-encoded GT5 trimers lower the precursor activation affinity threshold GT5 was engineered using our mammalian display bootstrapping approach, with GT mutations identified first against more mature-like antibodies and then modified to increase the immunogen's affinity for progressively more germline-like antibodies . The PCT64 LMCA sequence, isolated from the human donor early after infection, has some SHM in IGH-three aa mutations in the J gene and two aa from the full-length D gene ( Figure 7A). To test the capacity of the GT immunogens to activate more reverted germline IGHs, we generated a second mouse model where the J gene is fully reverted, PCT64 LMCA.JREVÀH (Figures S7A and S7B) (Willis et al., 2022). Genotyping and single-cell BCR sequencing confirmed that the PCT64 LMCA.JREVÀH sequence was expressed by $95% of the B cell repertoire ( Figure S7B). Through crossing with

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PCT64 LMCAÀL , we obtained mice where PCT64 LMCA.JREV IGH and IGK were paired in $84% of the repertoire ( Figure 7B). Reverting the PCT64 LMCA J-gene mutations decreased the affinity of PCT64 LMCA.JREV for GT2 (KD = 6.4 mM) and GT5 (KD = 347 nM). Antibodies with PCT64 LMCA.JREV IGH paired with murine IGK had a geomean affinity of 48.1 nM among binders, lower than the 16.7 nM geomean affinity of binders for PCT64 LMCA IGH paired with the same murine IGK ( Figure 7C). The lower affinity for PCT64 LMCA.JREV was reflected in the low frequencies of naive B cells binding GT2 (5%) and GT5 (55%) probes ( Figure 7D). To determine whether precursors in these affinity ranges could be triggered by immunization, PCT64 LMCA.JREVÀH B cells were transferred into CD45.1 WT mice (frequency: 100:10 6 ) prior to i.p.-immunization with either GT2 or GT5 protein trimers ( Figure S7C). GCs developed in response to both immunogens, but no PCT64 LMCA.JREVÀH B cell activation was detected after GT2 immunization at 8 dpi. However, PCT64 LMCA.JREV B cells were present in GCs after GT5 immunization ( Figure S7D), demonstrating that the improved GT5 immunogen can activate PCT64 precursors with LMCA.JREV BCRs. At 28 dpi, mutation frequency analysis identified enriched sites distinct from those induced in PCT64 LMCA B cells by GT5 ( Figure S7E). In particular, an aspartic acid (D) was acquired in position 117 in the HCDR3 in 98% of the GT5 + class-switched B cells ( Figure S7E); this mutation is present in the original PCT64 LMCA sequence ( Figure S7F), indicating a converging HCDR3-region maturation pathway.
The precursor frequency required for activation in PCT64 LMCA.JREV was far from the estimated human range. Therefore, we tested whether GT5-mRNA trimers could elicit stronger responses. We transferred PCT64 LMCA.JREV B cells (frequencies: 100, 20, and 10 per 10 6 ) and immunized recipient mice IM with 10 mg of mRNA-LNP encoding GT5 or 10 mg of GT5 soluble protein. At 13 dpi, GC responses with strong PCT64 LMCA.JREVÀH B cell recruitment were present in mRNAimmunized mice but no PCT64 LMCA.JREVÀH B cell activation was detected in protein-immunized mice ( Figure S7G). We then compared responses to mRNA-GT5 IM and GT5 protein i.p. immunization 13 and 42 dpi in inguinal LNs or spleen (Figure 7E). At 13 dpi CD45.2 PCT64 LMCA.JREV B cell responses to mRNA were 10-15 times higher than responses to protein ( Figures 7F and 7G). Furthermore, strong CD45.2 responses were present in the low frequency groups (20 and 10 per 10 6 ) ( Figures 7F and 7G). PCT64 LMCA.JREV GC responses to mRNA were maintained and expanded (reaching $20% of GC B cells) at 42 dpi in mice with a starting precursor frequency of 100 and 20 per 10 6 . Responses faded in mice with lower precursor numbers and in mice immunized with protein GT5 ( Figure 7H). Analysis of GT5 + class-switched B cells at 28 dpi revealed a mutation pattern overlapping with that induced by protein in JREV, characterized by an enrichment of Asp in position 117 in the HCDR3 ( Figure S7H).
These results suggest that mRNA-encoded GT5 may lower the affinity threshold for precursor activation Neuberger, 1998, 2000;Fleire et al., 2006). Thus, mRNA-LNP-encoded membrane-bound GT5 trimers may be promising candidates for the development of priming immunogens for PCT64-like precursors with diverse junctions and affinities.

DISCUSSION
Inducing bnAbs is a key objective in the quest for an HIV vaccine (Haynes and Burton, 2017). Central to these efforts is the structure-based design and validation of GT immunogens, which must activate rare precursor B cells, trigger durable GC responses, and induce desired mutations . Here, we engineered two BCR KI mice that express two different precursors of the V2-apex bnAb PCT64 to evaluate GT immunogens (Landais et al., 2017). In our preclinical models, high-affinity ApexGT5 was superior to ApexGT2 at activating rare PCT64 precursors. Furthermore, nucleoside-modified mRNA immunogens, at the relative doses administered, improved activation over protein.
BCR-immunogen affinity is a major determinant of rare B cell activation Dosenovic et al., 2018), and V2apex precursors are notably rare. Most HIV-1 bnAbs specific to V2-apex rely on HCDR3 loops with a 24-39 aa range to interact with their epitopes (Andrabi et al., 2015), and B cells bearing long HCDR3s (R24 aa) represent only 3.5% of the human repertoire (Briney et al. 2012). However, PCT64 precursors, with 25 aa HCDR3s, are estimated to have an upper frequency limit of 20 per 10 6 B cells. In contrast, 3.3 per 10 6 of B cells are eOD-GT8-specific VRC01 precursors   Article Havenar-Daughton et al., 2018;Lee et al., 2021). At the lowest precursor frequency investigated, the higher-affinity GT5 initiated strong, sustained activation with a 10-fold increase over GT2 in GC recruitment.
Both GT2 and GT5 elicited on-track SHM, induced reproducible HC mutation fingerprints, and did not display stringent LC restriction in our HC model, where various murine LCs pair with germline PCT64 IGHs. This suggests a predominantly IGHdependent selection process and validates the applicability of trimer design targeting HCDR3-dominant bnAb precursors previously tested only for BG18 (Steichen et al., 2019). There was, however, a marginal role for LCs: certain murine IGK pairings were so detrimental as to completely abrogate immunogen binding. In contrast, some advantageous IGK pairings increased the affinity of the original germline antibody. The capacity of a single immunogen to engage multiple B cell clones independently from the paired LC expands the range of precursors that can be elicited through immunization, raising the odds of developing bnAbs.
We also found that immunization with mRNA-LNP coding for membrane-bound GT5 was superior to soluble GT5 protein at triggering low-frequency PCT64-precursor B cells. This may be because V2-apex bnAbs tightly bind to quaternary epitopes, recognizing the N-linked glycan at residue 160 (N160) and interacting with a protein surface of the V2 domain of gp120 encompassing multiple protomers; they do not bind well to monomeric GP120 (Andrabi et al., 2015(Andrabi et al., , 2017Gift et al., 2017;Moore et al., 2017). IM immunization may negatively impact the successful presentation of an intact, fully-formed trimer structure; a similar effect has been previously reported in mice (Hu et al., 2015). Nucleoside-modified mRNA-LNP allows antigens to be translated into protein directly into the host's cells, minimizing processing by protease in antigen-presenting cells and increasing the chances that B cells encounter a well-folded trimer. Additionally, the amount of trimer produced after mRNA immunization may be larger than the protein dose, and antigen availability may be increased as a consequence of protein expression kinetics (Pardi et al., 2015(Pardi et al., , 2018Lederer et al., 2020). Indeed, similar responses are observed when immunogen administration is extended through an osmotic pump (Tam et al., 2016;Cirelli et al., 2019).
An HIV vaccine conferring broad protection will likely require the elicitation of several lines of antibodies to target multiple sites on Env. In this study, we validated HCDR3-focused GT immunogen design for the V2-apex epitope and demonstrated the utility of mRNA-LNP. As mRNA could simplify the delivery of multiple structurally sound immunogens, this work, alongside the array of GT vaccine candidates in various stages of clinical testing, may contribute to the development of a multi-component HIV vaccine.
Limitations of the study Our interpretation of SHM emphasized residues in the mature PCT64 bnAb; for some of those mutations, the precise role in binding remains to be clarified. Furthermore, it is not trivial to identify off-track mutations that might instead hinder bnAb development, particularly when they may only be detrimental in the context of other mutations.
While the PCT64-like mutations acquired in this study conferred autologous virus neutralization, booster immunizations with more native-like immunogens would likely be needed to achieve crossclade neutralization. Specifically, as R169 is rare among WT HIV isolates (<10%), it will be important to determine whether heterologous boost immunization can reduce the neutralization dependence on R169 observed. Finally, our conclusions have been derived from mouse models; further work will be required to see if they apply in non-human primates or humans.

STAR+METHODS
Detailed methods are provided in the online version of this paper and include the following:  Cryo-EM sample preparation Purified ApexGT2 or ApexGT5 was incubated overnight at 4 with $6 molar excess purified GT2-d42.16 or GT5-d42.16 Fab along with RM20A3 Fab then purified via size exclusion chromatography on a Superdex 200 Increase column followed by concentration of pooled fractions with a 30kD molecular weight cut-off Amicon Ultra centrifugal filter to a final concentration of $3-7 mg/ml. Concentrated sample was mixed with 0.5mL of 0.04 mM lauryl maltose neopentyl glycol (LMNG; Anatrace) to a final concentration of 0.005 mM and 4 mL of this solution was applied to plasma cleaned 1.2/1.3 C-Flat holey carbon grids (Electron Microscopy Sciences) using a Vitrobot mark IV (Thermo Fisher Scientific) with a 7 s blot time, 0 blot force, and wait time of 0 s. Prepared grids were then stored in liquid nitrogen until they were transfer to a microscope for imaging.

Cryo-EM data collection
A table of detailed imaging conditions and data statistics for all the EM datasets is presented in Table S1. All datasets were collected with Leginon automated microscopy software (Suloway et al., 2005) on either an FEI Titan Krios operating at 300keV or an FEI Talos Arctica operating at 200keV (Thermo Fisher Scientific), both equipped with a K2 Summit direct electron detector (Gatan) operated in counting mode.
Cryo-EM data processing All movie micrographs were aligned and dose-weighted using MotionCor2 (Zheng et al., 2017) and CTF parameters were estimated with GCTF (Zhang, 2016). Single-particle processing was carried out using a combination of Relion-3 (Kimanius et al., 2016;Zivanov et al., 2018) and CryoSparc2 (Punjani et al., 2017). The following general workflow was used for both datasets presented in this study. After frame alignment, dose-weighting, and CTF estimation, a subset of micrographs were selected based on CTF fit parameters and particle picking was performed, first using a gaussian blob, then templates from 2-D class averages. These particles were then subjected to one-two rounds of 2-D classification followed by subset selection, then one round of ab initio classification followed by subset. After subset selection, 3-Dautorefinement was performed with per-particle CTF correction followed by another round of 3-D classification using 3-D variability analysis. A soft spherical mask that surrounds the trimer apex and is large enough to accommodate the entire Fab was used to isolate variability in Fab occupancy followed by clustering into 3-6 classes. Clusters with clear density for Fab were then pooled and refined again together. 3-D variability was then employed again, this time to isolate variability in Fab binding angle followed by clustering and pooling of particles with similar angle of approach. Lastly, a final round of 3-D non-uniform refinement was performed to generate the final reconstruction.

Model building and figure preparation
For the GT2 + GT2-d42.16 Fab complex, the previously refined ApexGT2 (Willis et al., 2022) model was docked into the EM density map using UCSF Chimera (Pettersen et al., 2004) along with the previously refined atomic model of PCT64 LMCA and combined into a single PDB file. Mutations associated with GT5 relative to GT2 and d42.16 relative to the LMCA were then manually generated along with manual adjustment of glycans using COOT (Emsley and Crispin, 2018). This initial model was then relaxed into the EM density map using Rosetta  asking for $300 models. All models were validated using MolProbity (Chen et al., 2010) and EMRinger (Barad et al., 2015) and the model with the best combined score was selected. All models were then checked and adjusted manually in COOT and re-refined with Rosetta, if necessary. Final models were then scored again with MolProbity and EMRinger, while glycan structures were further validated with Privateer (Agirre et al., 2015). Figures were prepared with either UCSF Chimera or ChimeraX (Pettersen et al., 2021). Hydrogen bonds were calculated and displayed with UCSF ChimeraX. Volume segmentation was performed with Segger (Pintilie et al., 2010) as implemented in UCSF ChimeraX. Figures were prepared in Adobe Illustrator (Adobe Inc.) and PowerPoint (Microsoft).
For all mAb pseudovirus neutralization assays the IC50 (the concentration of mAb needed to obtain 50% neutralization against a given pseudovirus) was calculated from the non-linear regression of the neutralization curve. All neutralization assays were repeated at least twice, and data shown are from representative experiments.