mRNA-LNP HIV-1 trimer boosters elicit precursors to broad neutralizing antibodies.

Germline-targeting (GT) HIV vaccine strategies are predicated on deriving broadly neutralizing antibodies (bnAbs) through multiple boost immunogens. However, as the recruitment of memory B cells (MBCs) to germinal centers (GCs) is inefficient and may be derailed by serum antibody-induced epitope masking, driving further B cell receptor (BCR) modification in GC-experienced B cells after boosting poses a challenge. Using humanized immunoglobulin knockin mice, we found that GT protein trimer immunogen N332-GT5 could prime inferred-germline precursors to the V3-glycan-targeted bnAb BG18 and that B cells primed by N332-GT5 were effectively boosted by either of two novel protein immunogens designed to have minimum cross-reactivity with the off-target V1-binding responses. The delivery of the prime and boost immunogens as messenger RNA lipid nanoparticles (mRNA-LNPs) generated long-lasting GCs, somatic hypermutation, and affinity maturation and may be an effective tool in HIV vaccine development.

mRNA-LNP prime boost evolves precursors toward VRC01-like broadly neutralizing antibodies in preclinical humanized mouse models.

Germline-targeting (GT) protein immunogens to induce VRC01-class broadly neutralizing antibodies (bnAbs) to the CD4-binding site of the HIV envelope (Env) have shown promise in clinical trials. Here, we preclinically validated a lipid nanoparticle-encapsulated nucleoside mRNA (mRNA-LNP) encoding eOD-GT8 60mer as a soluble self-assembling nanoparticle in mouse models. In a model with three humanized B cell lineages bearing distinct VRC01-precursor B cell receptors (BCRs) with similar affinities for eOD-GT8, all lineages could be simultaneously primed and undergo diversification and affinity maturation without exclusionary competition. Boosts drove precursor B cell participation in germinal centers; the accumulation of somatic hypermutations, including in key VRC01-class positions; and affinity maturation to boost and native-like antigens in two of the three precursor lineages. We have preclinically validated a prime-boost regimen of soluble self-assembling nanoparticles encoded by mRNA-LNP, demonstrating that multiple lineages can be primed, boosted, and diversified along the bnAb pathway.

Eliciting a single amino acid change by vaccination generates antibody protection against group 1 and group 2 influenza A viruses.

Broadly neutralizing antibodies (bnAbs) targeting the hemagglutinin (HA) stem of influenza A viruses (IAVs) tend to be effective against either group 1 or group 2 viral diversity. In rarer cases, intergroup protective bnAbs can be generated by human antibody paratopes that accommodate the conserved glycan differences between the group 1 and group 2 stems. We applied germline-engaging nanoparticle immunogens to elicit a class of cross-group bnAbs from physiological precursor frequency within a humanized mouse model. Cross-group protection depended on the presence of the human bnAb precursors within the B cell repertoire, and the vaccine-expanded antibodies enriched for an N55T substitution in the CDRH2 loop, a hallmark of the bnAb class. Structurally, this single mutation introduced a flexible fulcrum to accommodate glycosylation differences and could alone enable cross-group protection. Thus, broad IAV immunity can be expanded from the germline repertoire via minimal antigenic input and an exceptionally simple antibody development pathway.

Microbial Inoculation during the Short-Term Composting Process Enhances the Nutritional and Functional Properties of Oyster Mushrooms (Pleurotus ostreatus).

In recent years, short-term composting techniques have been widely applied in oyster mushroom cultivation, but there is still a lack of systematic research on their impact on the nutritional and functional properties of fruiting bodies. In this study, the microbial inoculant Streptomyces thermoviolaceus BUA-FM01 (ST) was applied in the short-term composting process for oyster mushroom cultivation. The agronomic traits, nutritional composition, flavor compounds, and antioxidant activity of fruiting bodies from the first three flushes were evaluated. The results show that microbial inoculation significantly (p < 0.05) reduced the total carbon content and C/N ratio of the composted substrates and, furthermore, increased the total yield of the fruiting bodies. Moreover, microbial inoculation significantly (p < 0.05) increased the crude protein, crude polysaccharide, total amino acid, and essential amino acid contents of the fruiting bodies. The fruiting bodies of the first flush of ST treatment possessed the highest umami amino acid content and equivalent umami concentration value. Furthermore, microbial inoculation significantly (p < 0.05) enhanced the scavenging ability of crude polysaccharides toward free radicals. The results indicate that microbial inoculation has many benefits for the composting cultivating process of oyster mushrooms and good application prospects.

Focusing through scattering media by a single polarization transmission matrix with binary polarization modulation.

Wavefront distortion induced by scattering media seriously affects optical focusing. Wavefront shaping based on a transmission matrix (TM) is useful in controlling light propagation in highly scattering media. Traditional TM generally studies amplitude and phase, but the stochastic nature of the light propagation in the scattering medium also affects its polarization. Based on the binary polarization modulation, we propose a single polarization transmission matrix (SPTM) and achieve single-spot focusing through scattering media. We anticipate that the SPTM will be widely used in wavefront shaping.

Antibodies from primary humoral responses modulate the recruitment of naive B cells during secondary responses.

Vaccines generate high-affinity antibodies by recruiting antigen-specific B cells to germinal centers (GCs), but the mechanisms governing the recruitment to GCs on secondary challenges remain unclear. Here, using preclinical SARS-CoV and HIV mouse models, we demonstrated that the antibodies elicited during primary humoral responses shaped the naive B cell recruitment to GCs during secondary exposures. The antibodies from primary responses could either enhance or, conversely, restrict the GC participation of naive B cells: broad-binding, low-affinity, and low-titer antibodies enhanced recruitment, whereas, by contrast, the high titers of high-affinity, mono-epitope-specific antibodies attenuated cognate naive B cell recruitment. Thus, the directionality and intensity of that effect was determined by antibody concentration, affinity, and epitope specificity. Circulating antibodies can, therefore, be important determinants of antigen immunogenicity. Future vaccines may need to overcome-or could, alternatively, leverage-the effects of circulating primary antibodies on subsequent naive B cell recruitment.

Loss of Slc38a4 imprinting is a major cause of mouse placenta hyperplasia in somatic cell nuclear transferred embryos at late gestation.

Placenta hyperplasia is commonly observed in cloned animals and is believed to impede the proper development of cloned embryos. However, the mechanism underlying this phenomenon is largely unknown. Here, we show that placenta hyperplasia of cloned mouse embryos occurs in both middle and late gestation. Interestingly, restoring paternal-specific expression of an amino acid transporter Slc38a4, which loses maternal H3K27me3-dependent imprinting and becomes biallelically expressed in cloned placentae, rescues the overgrowth of cloned placentae at late gestation. Molecular analyses reveal that loss of Slc38a4 imprinting leads to over-activation of the mechanistic target of rapamycin complex 1 (mTORC1) signaling pathway in cloned placentae, which is likely due to the increased amino acids transport by SLC38A4. Collectively, our study not only reveals loss of Slc38a4 imprinting is responsible for overgrowth of cloned placentae at late gestation but also suggests the underlying mechanism involves increased amino acid transport and over-activation of mTORC1.

DPPA2 and DPPA4 are dispensable for mouse zygotic genome activation and pre-implantation development.

How maternal factors in oocytes initiate zygotic genome activation (ZGA) remains elusive in mammals, partly due to the challenge of de novo identification of key factors using scarce materials. Two-cell (2C)-like cells have been widely used as an in vitro model in order to understand mouse ZGA and totipotency because of their expression of a group of two-cell embryo-specific genes and their simplicity for genetic manipulation. Recent studies indicate that DPPA2 and DPPA4 are required for establishing the 2C-like state in mouse embryonic stem cells in a DUX-dependent manner. These results suggest that DPPA2 and DPPA4 are essential maternal factors that regulate Dux and ZGA in embryos. By analyzing maternal knockout and maternal-zygotic knockout embryos, we unexpectedly found that DPPA2 and DPPA4 are dispensable for Dux activation, ZGA and pre-implantation development. Our study suggests that 2C-like cells do not fully recapitulate two-cell embryos in terms of regulation of two-cell embryo-specific genes, and, therefore, caution should be taken when studying ZGA and totipotency using 2C-like cells as the model system.

Targeting lysophospholipid acid receptor 1 and ROCK kinases promotes antiviral innate immunity.

Growing evidence indicates the vital role of lipid metabolites in innate immunity. The lipid lysophosphatidic acid (LPA) concentrations are enhanced in patients upon HCV or SARS-CoV-2 infection, but the function of LPA and its receptors in innate immunity is largely unknown. Here, we found that viral infection promoted the G protein­coupled receptor LPA1 expression, and LPA restrained type I/III interferon production through LPA1. Mechanistically, LPA1 signaling activated ROCK1/2, which phosphorylated IRF3 Ser97 to suppress IRF3 activation. Targeting LPA1 or ROCK in macrophages, fibroblasts, epithelial cells, and LPA1 conditional KO mice promoted interferon-induced clearance of multiple viruses. LPA1 was colocalized with the receptor ACE2 in lung and intestine. Together with previous findings that LPA1 and ROCK1/2 promoted vascular leaking or lung fibrosis, we propose that the current available preclinical drugs targeting the LPA1-ROCK module might protect from SARS-CoV-2 or various virus infections in the intestine or lung.

Dosage effect of multiple genes accounts for multisystem disorder of myotonic dystrophy type 1.

Multisystem manifestations in myotonic dystrophy type 1 (DM1) may be due to dosage reduction in multiple genes induced by aberrant expansion of CTG repeats in DMPK, including DMPK, its neighboring genes (SIX5 or DMWD) and downstream MBNL1. However, direct evidence is lacking. Here, we develop a new strategy to generate mice carrying multigene heterozygous mutations to mimic dosage reduction in one step by injection of haploid embryonic stem cells with mutant Dmpk, Six5 and Mbnl1 into oocytes. The triple heterozygous mutant mice exhibit adult-onset DM1 phenotypes. With the additional mutation in Dmwd, the quadruple heterozygous mutant mice recapitulate many major manifestations in congenital DM1. Moreover, muscle stem cells in both models display reduced stemness, providing a unique model for screening small molecules for treatment of DM1. Our results suggest that the complex symptoms of DM1 result from the reduced dosage of multiple genes.

Stabilization of mouse haploid embryonic stem cells with combined kinase and signal modulation.

Mammalian haploid embryonic stem cells (haESCs) provide new possibilities for large-scale genetic screens because they bear only one copy of each chromosome. However, haESCs are prone to spontaneous diploidization through unknown mechanisms. Here, we report that a small molecule combination could restrain mouse haESCs from diploidization by impeding exit from naïve pluripotency and by shortening the S-G2/M phases. Combined with 2i and PD166285, our chemical cocktail could maintain haESCs in the haploid state for at least five weeks without fluorescence-activated cell sorting (FACS) enrichment of haploid cells. Taken together, we established an effective chemical approach for long-term maintenance of haESCs, and highlighted that proper cell cycle progression was critical for the maintenance of haploid state.

CRISPR-Cas9-Mediated Genetic Screening in Mice with Haploid Embryonic Stem Cells Carrying a Guide RNA Library.

Mouse androgenetic haploid embryonic stem cells (AG-haESCs) can support full-term development of semi-cloned (SC) embryos upon injection into MII oocytes and thus have potential applications in genetic modifications. However, the very low birth rate of SC pups limits practical use of this approach. Here, we show that AG-haESCs carrying deletions in the DMRs (differentially DNA methylated regions) controlling two paternally repressed imprinted genes, H19 and Gtl2, can efficiently support the generation of SC pups. Genetic manipulation of these DKO-AG-haESCs in vitro using CRISPR-Cas9 can produce SC mice carrying multiple modifications with high efficiency. Moreover, transfection of DKO-AG-haESCs with a constitutively expressed sgRNA library and Cas9 allows functional mutagenic screening. DKO-AG-haESCs are therefore an effective tool for the introduction of organism-wide mutations in mice in a single generation.

Correction of a genetic disease by CRISPR-Cas9-mediated gene editing in mouse spermatogonial stem cells.

Spermatogonial stem cells (SSCs) can produce numerous male gametes after transplantation into recipient testes, presenting a valuable approach for gene therapy and continuous production of gene-modified animals. However, successful genetic manipulation of SSCs has been limited, partially due to complexity and low efficiency of currently available genetic editing techniques. Here, we show that efficient genetic modifications can be introduced into SSCs using the CRISPR-Cas9 system. We used the CRISPR-Cas9 system to mutate an EGFP transgene or the endogenous Crygc gene in SCCs. The mutated SSCs underwent spermatogenesis after transplantation into the seminiferous tubules of infertile mouse testes. Round spermatids were generated and, after injection into mature oocytes, supported the production of heterozygous offspring displaying the corresponding mutant phenotypes. Furthermore, a disease-causing mutation in Crygc (Crygc(-/-)) that pre-existed in SSCs could be readily repaired by CRISPR-Cas9-induced nonhomologous end joining (NHEJ) or homology-directed repair (HDR), resulting in SSC lines carrying the corrected gene with no evidence of off-target modifications as shown by whole-genome sequencing. Fertilization using round spermatids generated from these lines gave rise to offspring with the corrected phenotype at an efficiency of 100%. Our results demonstrate efficient gene editing in mouse SSCs by the CRISPR-Cas9 system, and provide the proof of principle of curing a genetic disease via gene correction in SSCs.