Disease in the Pld4thss/thss Model of Murine Lupus Requires TLR9.

Phospholipase D4 (PLD4) is an endolysosomal exonuclease of ssRNA and ssDNA, rather than a phospholipase as its name suggests. Human polymorphisms in the PLD4 gene have been linked by genome-wide association studies to systemic sclerosis, rheumatoid arthritis, and systemic lupus erythematosus. However, B6.129 Pld4-/- mice develop features of a distinct disease, macrophage activation syndrome, which is reversed in mice mutated in TLR9. In this article, we compare a Pld4 null mutant identified on the BALB/c background, Pld4thss/thss, which has distinct phenotypes: short stature, thin hair, and features of systemic lupus erythematosus. All phenotypes analyzed were largely normalized in Pld4thss/thssTlr9-/- mice. Thus, Pld4thss/thss represents a rare model in which mouse lupus etiology is TLR9 dependent. Compared with PLD4-deficient B6 mice, Pld4thss/thss mice had elevated levels of serum IgG, IgG anti-dsDNA autoantibodies, BAFF, and IFN-γ and elevated B cell numbers. Overall, the data suggest that PLD4 deficiency can lead to a diverse array of rheumatological abnormalities depending upon background-modifying genes, and that these diseases of PLD4 deficiency are largely driven by TLR9 recognition of ssDNA.

Cleavage of DNA and RNA by PLD3 and PLD4 limits autoinflammatory triggering by multiple sensors.

Phospholipase D3 (PLD3) and PLD4 polymorphisms have been associated with several important inflammatory diseases. Here, we show that PLD3 and PLD4 digest ssRNA in addition to ssDNA as reported previously. Moreover, Pld3-/-Pld4-/- mice accumulate small ssRNAs and develop spontaneous fatal hemophagocytic lymphohistiocytosis (HLH) characterized by inflammatory liver damage and overproduction of Interferon (IFN)-γ. Pathology is rescued in Unc93b13d/3dPld3-/-Pld4-/- mice, which lack all endosomal TLR signaling; genetic codeficiency or antibody blockade of TLR9 or TLR7 ameliorates disease less effectively, suggesting that both RNA and DNA sensing by TLRs contributes to inflammation. IFN-γ made a minor contribution to pathology. Elevated type I IFN and some other remaining perturbations in Unc93b13d/3dPld3-/-Pld4-/- mice requires STING (Tmem173). Our results show that PLD3 and PLD4 regulate both endosomal TLR and cytoplasmic/STING nucleic acid sensing pathways and have implications for the treatment of nucleic acid-driven inflammatory disease.

Author Correction: Vaccine elicitation of HIV broadly neutralizing antibodies from engineered B cells.

A Correction to this paper has been published: https://doi.org/10.1038/s41467-020-20304-y.

Vaccine elicitation of HIV broadly neutralizing antibodies from engineered B cells.

HIV broadly neutralizing antibodies (bnAbs) can suppress viremia and protect against HIV infection. However, their elicitation is made difficult by low frequencies of appropriate precursor B cell receptors and the complex maturation pathways required to generate bnAbs from these precursors. Antibody genes can be engineered into B cells for expression as both a functional antigen receptor on cell surfaces and as secreted antibody. Here, we show that HIV bnAb-engineered primary mouse B cells can be adoptively transferred and vaccinated in immunocompetent mice resulting in the expansion of durable bnAb memory and long-lived plasma cells. Somatic hypermutation after immunization indicates that engineered cells have the capacity to respond to an evolving pathogen. These results encourage further exploration of engineered B cell vaccines as a strategy for durable elicitation of HIV bnAbs to protect against infection and as a contributor to a functional HIV cure.

B cells expressing authentic naive human VRC01-class BCRs can be recruited to germinal centers and affinity mature in multiple independent mouse models.

Animal models of human antigen-specific B cell receptors (BCRs) generally depend on "inferred germline" sequences, and thus their relationship to authentic naive human B cell BCR sequences and affinities is unclear. Here, BCR sequences from authentic naive human VRC01-class B cells from healthy human donors were selected for the generation of three BCR knockin mice. The BCRs span the physiological range of affinities found in humans, and use three different light chains (VK3-20, VK1-5, and VK1-33) found among subclasses of naive human VRC01-class B cells and HIV broadly neutralizing antibodies (bnAbs). The germline-targeting HIV immunogen eOD-GT8 60mer is currently in clinical trial as a candidate bnAb vaccine priming immunogen. To attempt to model human immune responses to the eOD-GT8 60mer, we tested each authentic naive human VRC01-class BCR mouse model under rare human physiological B cell precursor frequency conditions. B cells with high (HuGL18HL) or medium (HuGL17HL) affinity BCRs were primed, recruited to germinal centers, and they affinity matured, and formed memory B cells. Precursor frequency and affinity interdependently influenced responses. Taken together, these experiments utilizing authentic naive human VRC01-class BCRs validate a central tenet of germline-targeting vaccine design and extend the overall concept of the reverse vaccinology approach to vaccine development.

Induction of Cross-Reactive and Protective Antibody Responses After DNA Vaccination With MHCII-Targeted Stem Domain From Influenza Hemagglutinin.

Novel and more broadly protective vaccines against influenza are needed to efficiently meet antigenic drift and shift. Relevant to this end, the stem domain of hemagglutinin (HA) is highly conserved, and antibodies specific for epitopes located to the stem have been demonstrated to be able to confer broad protection against various influenza subtypes. However, a remaining challenge is to induce antibodies against the poorly immunogenic stem by vaccination strategies that can be scaled up for prophylactic vaccination of the general population. Here, we have developed DNA vaccines where the conserved stem domain of HA from influenza A/PR/8/34 (H1N1) and A/Shanghai/2/2013 (H7N9) was targeted toward MHC class II molecules on antigen-presenting cells (APC) for increased immunogenicity. Each of these vaccines induced antibodies that cross-reacted with other subtypes in the corresponding phylogenetic influenza groups. Importantly, when mixing the MHCII-targeted stem domains from H1N1 and H7N9 influenza viruses into one vaccine bolus, we observed broad protection against candidate stains from both phylogenetic groups 1 and 2.

PLD3 and PLD4 are single-stranded acid exonucleases that regulate endosomal nucleic-acid sensing.

The sensing of microbial genetic material by leukocytes often elicits beneficial pro-inflammatory cytokines, but dysregulated responses can cause severe pathogenesis. Genome-wide association studies have linked the gene encoding phospholipase D3 (PLD3) to Alzheimer's disease and have linked PLD4 to rheumatoid arthritis and systemic sclerosis. PLD3 and PLD4 are endolysosomal proteins whose functions are obscure. Here, PLD4-deficient mice were found to have an inflammatory disease, marked by elevated levels of interferon-γ (IFN-γ) and splenomegaly. These phenotypes were traced to altered responsiveness of PLD4-deficient dendritic cells to ligands of the single-stranded DNA sensor TLR9. Macrophages from PLD3-deficient mice also had exaggerated TLR9 responses. Although PLD4 and PLD3 were presumed to be phospholipases, we found that they are 5' exonucleases, probably identical to spleen phosphodiesterase, that break down TLR9 ligands. Mice deficient in both PLD3 and PLD4 developed lethal liver inflammation in early life, which indicates that both enzymes are needed to regulate inflammatory cytokine responses via the degradation of nucleic acids.

Design and crystal structure of a native-like HIV-1 envelope trimer that engages multiple broadly neutralizing antibody precursors in vivo.

Induction of broadly neutralizing antibodies (bNAbs) by HIV-1 envelope glycoprotein immunogens would be a major advance toward an effective vaccine. A critical step in this process is the activation of naive B cells expressing germline (gl) antibody precursors that have the potential to evolve into bNAbs. Here, we reengineered the BG505 SOSIP.664 glycoprotein to engage gl precursors of bNAbs that target either the trimer apex or the CD4-binding site. The resulting BG505 SOSIP.v4.1-GT1 trimer binds multiple bNAb gl precursors in vitro. Immunization experiments in knock-in mice expressing gl-VRC01 or gl-PGT121 show that this trimer activates B cells in vivo, resulting in the secretion of specific antibodies into the sera. A crystal structure of the gl-targeting trimer at 3.2-Å resolution in complex with neutralizing antibodies 35O22 and 9H+109L reveals a native-like conformation and the successful incorporation of design features associated with binding of multiple gl-bNAb precursors.

Tailored Immunogens Direct Affinity Maturation toward HIV Neutralizing Antibodies.

Induction of broadly neutralizing antibodies (bnAbs) is a primary goal of HIV vaccine development. VRC01-class bnAbs are important vaccine leads because their precursor B cells targeted by an engineered priming immunogen are relatively common among humans. This priming immunogen has demonstrated the ability to initiate a bnAb response in animal models, but recall and maturation toward bnAb development has not been shown. Here, we report the development of boosting immunogens designed to guide the genetic and functional maturation of previously primed VRC01-class precursors. Boosting a transgenic mouse model expressing germline VRC01 heavy chains produced broad neutralization of near-native isolates (N276A) and weak neutralization of fully native HIV. Functional and genetic characteristics indicate that the boosted mAbs are consistent with partially mature VRC01-class antibodies and place them on a maturation trajectory that leads toward mature VRC01-class bnAbs. The results show how reductionist sequential immunization can guide maturation of HIV bnAb responses.