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We report the identification of a new recessive prenatal lethal insertional mutation, amnionless (amn). amn mutant embryos first appear abnormal during the Early Streak stage, between E6.5 and E7.0, when they initiate mesoderm production. Subsequently, the amn mutants become developmentally arrested between the Mid and Late Streak stages of gastrulation and they die and are resorbed between E9.5 and E10.5. While extraembryonic structures, including the chorion, yolk sac blood islands, and allantois appear to develop normally, the small embryonic ectoderm remains undifferentiated and generates no amnion. In addition, the embryonic mesoderm that is produced does not become organized into node, notochord, and somites and there is no morphological evidence of neural induction. Interspecific backcross and fluorescence in situ hybridization analyses map the transgene insertion, and thus the amn mutation, to the distal region of mouse chromosome 12, which has synteny with human chromosome 14q32. A gene encoding a 7.5-kb transcript has been identified at a junction between the integrated transgene and host chromosome 12 sequences that meets three criteria expected of a candidate amn gene. This gene maps to the site of transgene insertion; it is transcribed during gastrulation, and its expression is disrupted in amn mutant embryos. Nucleotide sequencing studies show that the 567 amino acid protein encoded by the 7.5-kb transcript is a member of the newly defined family of putative signal transducing proteins, TRAFs, that associate with the cytoplasmic domains of members of the tumor necrosis factor (TNF) receptor superfamily. Thus, we have named the gene encoding the 7.5-kb transcript TRAFamn. TRAFamn is identical to a recently reported protein (CD40bp, CAP-1, CRAF1, LAP1) that can bind the cytoplasmic domains of CD40 and the lymphotoxin [Beta] receptor (LT[Beta]R), both of which are known members of the TNF receptor superfamily. The implications of these findings regarding a possible role for the TNF receptor superfamily during gastrulation are discussed.

The ligand-binding surface of the B cell receptor (BCR) is formed by encoded and non-encoded antigen complementarity determining regions (CDRs). Genetically reproducible or 'public' antibodies can arise when the encoded CDRs play deterministic roles in antigen recognition, notably within human broadly neutralizing antibodies against HIV and influenza virus. We sought to exploit this by engineering virus-like-particle (VLP) vaccines that harbor multivalent affinity against gene-encoded moieties of the BCR antigen binding site. As proof of concept, we deployed a library of RNA bacteriophage VLPs displaying random peptides to identify a multivalent antigen that selectively triggered germline BCRs using the human VH gene IGVH1-2*02. This VLP selectively primed IGHV1-2*02 BCRs that were present within a highly diversified germline antibody repertoire within humanized mice. Our approach thus provides methodology to generate antigens that engage specific BCR configurations of interest, in the absence of structure-based information. [ABSTRACT FROM AUTHOR]

Studies on transgenic mice that express human IgM, IgG and Igkappa reveal that human immunoglobulin-expressing B cells develop and respond normally in a mouse immune system. The antigen response causes immunoglobulin heavy-chain isotype switching and differential somatic mutations. The use of conventional hybridoma technology enables the acquisition of human sequence antibodies from these mice.

The analysis of the expression of a human immunoglobulin heavy-chain minilocus in transgenic mice was reported. The transgenic minilocus is expressed in mice in a manner analogous to that of the human heavy-chain complex. The repertoire was fetal in appearance with the absence of somatic mutation. It was found that the two heavy-chain variable gene segments rearranged to micro- and gamma-chain constant segments. The functional gene segment is rearranged more frequently than the pseudogene. Thus, it was shown that the functional gene segment can undergo a class-switch.

Laboratory mice provide a ready source of diverse, high-affinity and high-specificity monoclonal antibodies (mAbs). However, development of rodent antibodies as therapeutic agents has been impaired by the inherent immunogenicity of these molecules. One technology that has been explored to generate low immunogenicity mAbs for in vivo therapy involves the use of transgenic mice expressing repertoires of human antibody gene sequences. This technology has now been exploited by over a dozen different pharmaceutical and biotechnology companies toward developing new therapeutic mAbs, and currently at least 33 different drugs in clinical testing—including several in pivotal trials—contain variable regions encoded by human sequences from transgenic mice. The emerging data from these trials provide an early glimpse of the safety and efficacy issues for these molecules. Nevertheless, actual product approval, the biggest challenge so far, is required to fully validate this technology as a drug discovery tool. In the future, it may be possible to extend this technology beyond rodents and use transgenic farm animals to directly generate and produce human sequence polyclonal sera. [ABSTRACT FROM AUTHOR]

Reports on the use of mice for obtaining high-affinity human antibodies. Mice with human sequence transgenes undergoing V(D)J mutant strain and heavy-chain class switching; Use of somatic mutation to generate a repertoire of human sequence immunoglobulins; Potential as therapeutic agents of human sequence monoclonal antibodies.

We have reported previously that mice carrying >30 copies of the human CD3ε transgene completely lose their T lymphocytes and NK cells (36). Here we demonstrate by immunohistology that in the most severely immunodeficient mouse, tgε26, the thymus is very small, has sizeable vacuoles and does not contain recognizable T lymphocytes except for a small percentage of Thy- 1 cells and B cells. Cell surface phenotyping and TCRα and -β rearrangement studies confirm that the arrest in T lymphocyte development precedes the arrest in , and TCRβ mice. Since the T cell progenitors in which the arrest occurred were absent in the transgenic mice, indirect approaches were taken to examine the causes of the block in T cell development. Analyses of 12 independently established mutant mouse lines, generated with five different transgenic constructs, revealed that the severity of the abrogation in T cell development was dependent on the number of copies of transgenes. Since the number of transgene copies generally correlated with the levels of expression of the transgenic CD3ε proteins, we concluded that over-expression of the CD3ε protein was the likely cause of the block in T lymphocyte development. The T cell immunodeficiency was caused by either the human or the murine CD3ε protein. Since transgene coded mRNAs were found in significantly higher quantities than endogenous CD3ε mRNAs in fetal thymi on days 13 and 14 of gestation, over-expression took place very early in development, probably prematurely. Over-expression of the CD3ε transgene in thymocyte precursors may therefore affect T lymphocyte development in the absence of TCR and possibly in the absence of the other CD3 proteins. More importantly, over-expression of the CD3ε protein in thymocytes of mice with a low copy number of transgenes had a significant effect on late thymic development Over-expression of the CD3ε protein in immature thymocytes mimicked the effects caused by exposure of CD4; CD8 thymocytes to anti-CD3ε treatment: apoptosis and lack of TCRβ expression. We therefore speculate that in the homozygous tgε26 animals the arrest in T cell development was caused by excessive signal transduction events rather than by a toxic effect of the transgenic protein. [ABSTRACT FROM PUBLISHER]

We have generated transgenic mice that contain human-sequence Ig mlnllocl and, because they are also homozygous for a targeted disruption of their endogenous heavy chain genes, must rely on the transgene sequences for B cell receptor expression. Although the human transgenes contain only a fraction of the intact human heavy chain locus, these defined sequences are able to at least partially restore the humoral immune system in the mouse. B cells expressing human heavy chains develop in the bone marrow, populate peripheral lymphoid tissue and respond specifically to antigen. Furthermore, the heavy chain transgenes contain both human μ and γ1 coding exons as well as the respective μ and γ1 switch regions. The sequences included within the transgene are sufficient to direct class switch recombination. Transgene sequences are also sufficient to direct somatic mutation of the class-switched heavy chain genes. These observations define the upper limit of the c/s-actlng sequences necessary to direct heavy chain class switching and somatic mutation. [ABSTRACT FROM PUBLISHER]

B cell receptors (BCRs) display a combination of variable (V)-gene-encoded complementarity determining regions (CDRs) and adaptive/hypervariable CDR3 loops to engage antigens. It has long been proposed that the former tune for recognition of pathogens or groups of pathogens. To experimentally evaluate this within the human antibody repertoire, we perform immune challenges in transgenic mice that bear diverse human CDR3 and light chains but are constrained to different human V H - genes. We find that, of six commonly deployed V H sequences, only those CDRs encoded by IGHV1-2∗02 enable polyclonal antibody responses against bacterial lipopolysaccharide (LPS) when introduced to the bloodstream. The LPS is from diverse strains of gram-negative bacteria, and the V H -gene-dependent responses are directed against the non-variable and universal saccrolipid substructure of this antigen. This reveals a broad-spectrum anti-LPS response in which germline-encoded CDRs naturally hardwire the human antibody repertoire for recognition of a conserved microbial target. • Transgenic mice alter V H -gene contribution to human-like BCR repertoires • Specific human V H -genes are needed to engage bloodborne bacterial LPS in vivo • Germline-endowed antigen recognition targets a universally conserved LPS moiety The ligand binding surface of antibodies is generated by diverse gene-encoded and hypervariable loops. Sangesland et al. demonstrate that certain gene-encoded loops are needed for human antibodies to accommodate the shapes of some bacterial antigens and naturally tune for recognition of conserved microbial features therein. [ABSTRACT FROM AUTHOR]

Human broadly neutralizing antibodies (bnAbs) targeting the hemagglutinin stalk of group 1 influenza A viruses (IAVs) are biased for IGHV1-69 alleles that use phenylalanine (F54) but not leucine (L54) within their CDRH2 loops. Despite this, we demonstrated that both alleles encode for human IAV bnAbs that employ structurally convergent modes of contact to the same epitope. To resolve differences in lineage expandability, we compared F54 versus L54 as substrate within humanized mice, where antibodies develop with human-like CDRH3 diversity but are restricted to single V H genes. While both alleles encoded for bnAb precursors, only F54 IGHV1-69 supported elicitation of heterosubtypic serum bnAbs following immunization with a stalk-only nanoparticle vaccine. L54 IGHV1-69 was unproductive, co-encoding for anergic B cells and autoreactive stalk antibodies that were cleared from B cell memory. Moreover, human stalk antibodies also demonstrated L54-dependent autoreactivity. Therefore, IGHV1-69 polymorphism, which is skewed ethnically, gates tolerance and vaccine expandability of influenza bnAbs. [Display omitted] • Both F54 and L54 IGHV1-69 alleles encode for human bnAbs against influenza virus • Only F54 IGHV1-69 can provide a substrate for vaccine-amplifying bnAbs to high titer • L54 IGHV1-69 co-endows for poly/autoreactive B cells, which become tolerized • Immune cell checkpoint inhibitors can restore L54 IGHV1-69 antibody responses Human broadly neutralizing antibodies (bnAbs) against the influenza virus preferentially use F54 IGHV1-69 as opposed to the L54 allele of this antibody V H gene. Sangesland et al. show that although both alleles encode for affinity to the same target, L54 IGHV1-69 also imparts autoreactivity, dampening vaccine expandability in a humanized mouse model. [ABSTRACT FROM AUTHOR]

Antibody paratopes are formed by hypervariable complementarity-determining regions (CDRH3s) and variable gene-encoded CDRs. The latter show biased usage in human broadly neutralizing antibodies (bnAbs) against both HIV and influenza virus, suggesting the existence of gene-endowed targeting solutions that may be amenable to pathway amplification. To test this, we generated transgenic mice with human CDRH3 diversity but simultaneously constrained to individual user-defined human immunoglobulin variable heavy-chain (V H) genes, including IGHV1-69, which shows biased usage in human bnAbs targeting the hemagglutinin stalk of group 1 influenza A viruses. Sequential immunization with a stalk-only hemagglutinin nanoparticle elicited group 1 bnAbs, but only in IGHV1-69 mice. This V H -endowed response required minimal affinity maturation, was elicited alongside pre-existing influenza immunity, and when IGHV1-69 B cells were diluted to match the frequency measured in humans. These results indicate that the human repertoire could, in principle, support germline-encoded bnAb elicitation using a single recombinant hemagglutinin immunogen. • Generated mice with user-defined human antibody V H genes and humanized CDRH3 diversity • IGHV1-69 use enables elicitation of IgG against a "universal" site on influenza • A single flu immunogen elicited gene-encoded broadly neutralizing antibodies • The response was achieved when IGHV1-69 B cells were titrated to human frequency Human broadly neutralizing antibodies (bnAbs) against influenza virus can be biased for V H gene usage, suggesting gene-encoded development pathways. Sangesland et al. show that human IGHV1-69 B cell receptors impart natural affinity for a "universal" vaccine target, enabling rapid bnAb responses in mice that were elicited using a rationally designed influenza immunogen. [ABSTRACT FROM AUTHOR]