Human Ig knockin mice to study the development and regulation of HIV-1 broadly neutralizing antibodies

Abstract

A major challenge for HIV-1 vaccine research is developing a successful immunization approach for inducing broadly neutralizing antibodies (bnAbs). A key shortcoming in meeting this challenge has been the lack of animal models capable of identifying impediments limiting bnAb induction and ranking vaccine strategies for their ability to promote bnAb development. Since 2010, immunoglobulin knockin (KI) technology, involving inserting functional rearranged human variable exons into the mouse IgH and IgL loci has been used to express bnAbs in mice. This approach has allowed immune tolerance mechanisms limiting bnAb production to be elucidated and strategies to overcome such limitations to be evaluated. From these studies, along with the wealth of knowledge afforded by analyses of recombinant Ig-based bnAb structures, it became apparent that key functional features of bnAbs often are problematic for their elicitation in mice by classic vaccine paradigms, necessitating more iterative testing of new vaccine concepts. In this regard, bnAb KI models expressing deduced precursor V(D)J rearrangements of mature bnAbs or unrearranged germline V, D, J segments (that can be assembled into variable region exons that encode bnAb precursors), have been engineered to evaluate novel immunogens/regimens for effectiveness in driving bnAb responses. One promising approach emerging from such studies is the ability of sequentially administered, modified immunogens (designed to bind progressively more mature bnAb precursors) to initiate affinity maturation. Here, we review insights gained from bnAb KI studies regarding the regulation and induction of bnAbs, and discuss new Ig KI methodologies to manipulate the production and/or expression of bnAbs in vivo, to further facilitate vaccine-guided bnAb induction studies.

Keywords: B-cell lineages; B-cell tolerance controls; CD4-binding site (CD4bs); broadly neutralizing antibodies (bnAbs); knockin (KI) mice; sequential immunization.

Fig 1. B-cell tolerance controls in the 2F5 bnAb KI mouse model

Shown is a pictorial summary of all bnAb regulation and induction studies done in the setting of 2F5 V_HDJ_H^+/++VκJκ^+/+ KI mice. The original (affinity matured) version of 2F5, upon knocking its VDJ/VJ rearrangements into B-cells, fails to pass multiple tolerance checkpoints (–85). However, while nearly all 2F5⁺ B-cells are clonally deleted at the 1st checkpoint (the pre->imm. B transition), 5% populate the periphery, but most have been functionally silenced. Amongst these residual anergic B-cells, a minority remove all self-reactivity & escape tolerance altogether, but due to the inability of 2F5 receptor editing to remove dominant 2F5 HC self-reactivity, purifying selection occurs in later development, by a T-dependent, SHM-driven process, “Affinity Reversion/Ab Redemption” (23, 30, 31, 37). Finally, serum bnAb IgG responses can be elicited in 2F5 KI mice immunized with an MPER peptide-lipid-TLR agonist vaccine regime through re-activation of unedited (MPER⁺) anergic KI B cells not yet subjected to purifying selection (84, 85).

Fig 2. Mouse KI/chimeric models for testing VRC01 class-targeting vaccine strategies

Schematic illustration of VRC01gH, 3BNC60 GLV_H, 3BNC60 MuV_H, or gl-3BNC60 HC/LC mouse KI and V_H1-2 or V_H1-2/LC chimeric model alleles generated to evaluate the ability of CD4bs-targeting sequential immunization strategies to induce VRC01-like Abs.

Fig 2. Mouse KI/chimeric models for testing VRC01 class-targeting vaccine strategies

Schematic illustration of VRC01gH, 3BNC60 GLV_H, 3BNC60 MuV_H, or gl-3BNC60 HC/LC mouse KI and V_H1-2 or V_H1-2/LC chimeric model alleles generated to evaluate the ability of CD4bs-targeting sequential immunization strategies to induce VRC01-like Abs.

Fig 3. PGT121-class bnAb KI models

Schematic illustration of PGT121 GL_HL and PGT121 Mut_HGL_L KI alleles generated to evaluate the ability of V3-targeting sequential immunization strategies to induce PGT121-like Abs.

Fig 4. Types of Ig-humanized mouse models to study vaccine-guided bnAb development

Pictorial representations of the structural organization of targeted alleles from various kinds of current or planned Ig-humanized models are shown, relative to the WT murine allele. For simplicity, only HC loci are depicted, and only at one allele. Models that have been published (with corresponding references to the right), or models which are being generated, are denoted in black text (Models A, B, and E), while theoretical germline/unrearranged models (that could be made to further bridge the current gap between physiological relevance and practicality to study “difficult to infer, long HCDR3” bnAb specificities; Models C and D), are annotated blue text. Mouse and human germline-derived sequences/segments are depicted in red and blue, respectively. Denoted as a green segment in the A1 type of models are “mutated” HCDR3s from the human bnAbs VRC01 and PGT121, i.e. from either the original/mature VRC01 bnAb, but with a single mutation to remove an unpaired cysteine (118), or for PGT121, from the least mutated lineage member (132). The intergenic control region 1 (IGCR1) normally found in WT HC loci is denoted by a large circle, which has been removed in models B,C, D, to further facilitate rearrangement of D-proximal (V_H81X-positioned) V_H segment(s) of interest. p=promoter region; L=leader sequence.

Fig. 5. High-throughput in vivo expression of bnAbs in chimeric mice using RDBC

Shown are the two major steps involved in generating bnAb KI chimeric mice using RAG-dependent blastocyst complementation: generation of blastocyst chimeras by microinjection of bnAb KI ES cells (A), and implantation of blastocysts in pseudopregnant RAG2^−/− foster mothers, resulting in chimeric offspring (B) whose lymphocytes all express the knocked-in bnAb specificity and can be used for immunization. Shown as an optional step is germline transmission of bnAb KI allele to generate a standard KI model (C), which can be pursued if more in-depth studies with a particular model are desired.