The histone demethylase KDM5C controls female bone mass by promoting energy metabolism in osteoclasts.

Women experience osteoporosis at higher rates than men. Aside from hormones, the mechanisms driving sex-dependent bone mass regulation are not well understood. Here, we demonstrate that the X-linked H3K4me2/3 demethylase KDM5C regulates sex-specific bone mass. Loss of KDM5C in hematopoietic stem cells or bone marrow monocytes increases bone mass in female but not male mice. Mechanistically, loss of KDM5C impairs the bioenergetic metabolism, resulting in impaired osteoclastogenesis. Treatment with the KDM5 inhibitor reduces osteoclastogenesis and energy metabolism of both female mice and human monocytes. Our report details a sex-dependent mechanism for bone homeostasis, connecting epigenetic regulation to osteoclast metabolism and positions KDM5C as a potential target for future treatment of osteoporosis in women.

The histone demethylase KDM5C controls female bone mass by promoting energy metabolism in osteoclasts.

Women experience osteoporosis at higher rates than men. Aside from hormones, the mechanisms driving sex-dependent bone mass regulation are not well-understood. Here, we demonstrate that the X-linked H3K4me2/3 demethylase KDM5C regulates sex-specific bone mass. Loss of KDM5C in hematopoietic stem cells or bone marrow monocytes (BMM) increases bone mass in female but not male mice. Mechanistically, loss of KDM5C impairs the bioenergetic metabolism resulting in impaired osteoclastogenesis. Treatment with the KDM5 inhibitor reduces osteoclastogenesis and energy metabolism of both female mice and human monocytes. Our report details a novel sex-dependent mechanism for bone homeostasis, connecting epigenetic regulation to osteoclast metabolism, and positions KDM5C as a target for future treatment of osteoporosis in women. One-Sentence Summary: KDM5C, an X-linked epigenetic regulator, controls female bone homeostasis by promoting energy metabolism in osteoclasts.

TRIM28 secures skeletal stem cell fate during skeletogenesis by silencing neural gene expression and repressing GREM1/AKT/mTOR signaling axis.

Long bones are generated by mesoderm-derived skeletal progenitor/stem cells (SSCs) through endochondral ossification, a process of sequential chondrogenic and osteogenic differentiation tightly controlled by the synergy between intrinsic and microenvironment cues. Here, we report that loss of TRIM28, a transcriptional corepressor, in mesoderm-derived cells expands the SSC pool, weakens SSC osteochondrogenic potential, and endows SSCs with properties of ectoderm-derived neural crest cells (NCCs), leading to severe defects of skeletogenesis. TRIM28 preferentially enhances H3K9 trimethylation and DNA methylation on chromatin regions more accessible in NCCs; loss of this silencing upregulates neural gene expression and enhances neurogenic potential. Moreover, TRIM28 loss causes hyperexpression of GREM1, which is an extracellular signaling factor promoting SSC self-renewal and SSC neurogenic potential by activating AKT/mTORC1 signaling. Our results suggest that TRIM28-mediated chromatin silencing establishes a barrier for maintaining the SSC lineage trajectory and preventing a transition to ectodermal fate by regulating both intrinsic and microenvironment cues.

Skip the buffet, for SPARC's sake.

Caloric restriction (CR) reduces inflammation and the incidence of chronic diseases, thereby extending healthspan and lifespan. In this issue of Immunity, Ryu et al. (2022) propose that reduction of SPARC, a matricellular protein, during CR offers beneficial effects by reducing SPARC-driven inflammatory phenotypes in macrophages.

Novel expression of coat proteins from thermophilic bacteriophage ΦIN93 and evaluation for assembly into virus-like particles.

Virus-like particles (VLPs) have the potential to be used as display platforms to develop vaccines against infectious and non-infectious agents. However, most VLPs used as vaccine display platforms are derived from viruses that infect humans; unfortunately, most humans already have pre-existing antibodies against these platforms and thus, the immunogenicity of these vaccines may be compromised. VLP platforms derived from viruses that infect bacteria (bacteriophages), especially bacteriophages that infect bacteria, which do not colonize humans are less likely to have pre-existing antibodies against the platforms in the human population. In this study, we assessed whether two putative coat proteins (ORF13 and ORF14) derived from a thermophilic bacteriophage (ΦIN93) can be expressed and purified from a mesophilic bacterium such as E. coli. We also assessed whether expressed coat proteins can assemble to form VLPs. Truncated versions of ORF13 and ORF14 were successfully co-expressed in bacteria; the co-expressed truncated proteins formed oval structures that look like VLPs, but their sizes were less than those of an authentic ΦIN93 virus.

Mixed Bacteriophage MS2-L2 VLPs Elicit Long-Lasting Protective Antibodies against HPV Pseudovirus 51.

Three prophylactic vaccines are approved to protect against HPV infections. These vaccines are highly immunogenic. The most recent HPV vaccine, Gardasil-9, protects against HPV types associated with ~90% of cervical cancer (worldwide). Thus, ~10% of HPV-associated cancers are not protected by Gardasil-9. Although this is not a large percentage overall, the HPV types associated with 10% of cervical cancer not protected by the current vaccine are significantly important, especially in HIV/AIDS patients who are infected with multiple HPV types. To broaden the spectrum of protection against HPV infections, we developed mixed MS2-L2 VLPs (MS2-31L2/16L2 VLPs and MS2-consL2 (69-86) VLPs) in a previous study. Immunization with the VLPs neutralized/protected mice against infection with eleven high-risk HPV types associated with ~95% of cervical cancer and against one low-risk HPV type associated with ~36% of genital warts & up to 32% of recurrent respiratory papillomatosis. Here, we report that the mixed MS2-L2 VLPs can protect mice from three additional HPV types: HPV51, which is associated with ~0.8% of cervical cancer; HPV6, which is associated with up to 60% of genital warts; HPV5, which is associated with skin cancers in patients with epidermodysplasia verruciformis (EV). Overall, mixed MS2-L2 VLPs can protect against twelve HPV types associated with ~95.8% of cervical cancers and against two HPV types associated with ~90% of genital warts and >90% recurrent respiratory papillomatosis. Additionally, the VLPs protect against one of two HPV types associated with ~90% of HPV-associated skin cancers in patients with EV. More importantly, we observed that mixed MS2-L2 VLPs elicit protective antibodies that last over 9 months. Furthermore, a spray-freeze-dried formulation of the VLPs is stable, immunogenic, and protective at room temperature and 37 °C.

Bacteriophage Qß virus-like particles displaying Chikungunya virus B-cell epitopes elicit high-titer E2 protein antibodies but fail to neutralize a Thailand strain of Chikungunya virus.

Chikungunya virus (CHIKV) is a mosquito-borne virus associated with arthritis and musculoskeletal pains. More than 2.9 million people worldwide have been infected with the virus within the last 1.5 decades; currently, there are no approved vaccines to protect against CHIKV infection. To assess the potential of using CHIKV peptides as vaccine antigens, we multivalently displayed CHIKV peptides representing B-cell epitopes (amino acids 2800-2818, 3025-3058, 3073-3081, 3121-3146, and 3177-3210), from E2 glycoprotein (Singapore strain), on the surface of a highly immunogenic bacteriophage Qß virus-like particle (VLP). We assessed the immunogenicity of CHIKV E2 amino acid 3025-3058 (including the other epitopes) displayed on Qß VLPs in comparison to the same peptide not displayed on VLPs. Mice immunized with the E2 peptides displayed on Qß VLPs elicited high-titer antibodies compared with the group immunized just with the peptide. However, sera from immunized mice did not neutralize CHIKV AF15561 (isolated from Thailand). The data suggest that Qß VLPs is an excellent approach to elicit high-titer CHIKV E2-protein antibodies at a lower dose of antigen and future studies should assess whether Qß-CHIKV E2 aa 2800-2818 VLPs and Qß-CHIKV E2 aa 3025-3058 VLPs can neutralize a Singapore Strain of CHIKV.

Virus-like Particle-Based L2 Vaccines against HPVs: Where Are We Today?

Human papillomaviruses (HPVs) are the most common sexually transmitted infections worldwide. Ninety percent of infected individuals clear the infection within two years; however, in the remaining 10% of infected individuals, the infection(s) persists and ultimately leads to cancers (anogenital cancers and head and neck cancers) and genital warts. Fortunately, three prophylactic vaccines have been approved to protect against HPV infections. The most recent HPV vaccine, Gardasil-9 (a nonavalent vaccine), protects against seven HPV types associated with ~90% of cervical cancer and against two HPV types associated with ~90% genital warts with little cross-protection against non-vaccine HPV types. The current vaccines are based on virus-like particles (VLPs) derived from the major capsid protein, L1. The L1 protein is not conserved among HPV types. The minor capsid protein, L2, on the other hand, is highly conserved among HPV types and has been an alternative target antigen, for over two decades, to develop a broadly protective HPV vaccine. The L2 protein, unlike the L1, cannot form VLPs and as such, it is less immunogenic. This review summarizes current studies aimed at developing HPV L2 vaccines by multivalently displaying L2 peptides on VLPs derived from bacteriophages and eukaryotic viruses. Recent data show that a monovalent HPV L1 VLP as well as bivalent MS2 VLPs displaying HPV L2 peptides (representing amino acids 17-36 and/or consensus amino acids 69-86) elicit robust broadly protective antibodies against diverse HPV types (6/11/16/18/26/31/33/34/35/39/43/44/45/51/52/53/56/58/59/66/68/73) associated with cancers and genital warts. Thus, VLP-based L2 vaccines look promising and may be favorable, in the near future, over current L1-based HPV vaccines and should be explored further.

Oral immunization with bacteriophage MS2-L2 VLPs protects against oral and genital infection with multiple HPV types associated with head & neck cancers and cervical cancer.

Human papillomaviruses (HPVs) are the most common sexually transmitted infections. HPVs are transmitted through anogenital sex or oral sex. Anogenital transmission/infection is associated with anogenital cancers and genital warts while oral transmission/infection is associated with head and neck cancers (HNCs) including recurrent respiratory papillomatosis. Current HPV vaccines protect against HPV types associated with ∼90% of cervical cancers and are expected to protect against a percentage of HNCs. However, only a few studies have assessed the efficacy of current vaccines against oral HPV infections. We had previously developed a mixed MS2-L2 candidate HPV vaccine based on bacteriophage MS2 virus-like particles (VLPs). The mixed MS2-L2 VLPs consisted of a mixture of two MS2-L2 VLPs displaying: i) a concatemer of L2 peptide (epitope 20-31) from HPV31 & L2 peptide (epitope 17-31) from HPV16 and ii) a consensus L2 peptide representing epitope 69-86. The mixed MS2-L2 VLPs neutralized/protected mice against six HPV types associated with ∼87% of cervical cancer. Here, we show that the mixed MS2-L2 VLPs can protect mice against additional HPV types; at the genital region, the VLPs protect against HPV53, 56, 11 and at the oral region, the VLPs protect against HPV16, 35, 39, 52, and 58. Thus, mixed MS2-L2 VLPs protect against eleven oncogenic HPV types associated with ∼95% of cervical cancer. The VLPs also have the potential to protect, orally, against the same oncogenic HPVs, associated with ∼99% of HNCs, including HPV11, which is associated with up to 32% of recurrent respiratory papillomatosis. Moreover, mixed MS2-L2 VLPs are thermostable at room temperature for up to 60 days after spray-freeze drying and they are protective against oral HPV infection.

Evaluation of the thermal stability and the protective efficacy of spray-dried HPV vaccine, Gardasil® 9.

High-risk human papillomavirus (HPV) types are responsible for nearly all cases of cervical cancers. Cervarix® and Gardasil® 9 are the current prophylactic vaccines available that protect against the majority of HPVs associated with cancer. Although these vaccines are highly effective, HPV vaccine implementation has been slow, particularly in low-and-middle income countries. Major barriers to the widespread availability of the HPV vaccines is its cost and the requirement for continuous refrigeration (2-8°C). Here, we used spray drying along with stabilizing excipients to formulate a thermostable Gardasil® 9 vaccine. We evaluated the immunogenicity and protective efficacy of the vaccine in mice immediately after spray drying and following storage for three months at 4°C, 25°C, and 40°C. The immunogenicity studies were performed using Gardasil® 9 as a whole antigen, and not individual HPV types, for ELISA. At the dose tested, the spray dried vaccine conferred protection against HPV following storage at temperatures up to 40°C. In addition to the spray-dried vaccine, our studies revealed that the Gardasil® 9 vaccine, as currently marketed, may be stored and transported at elevated temperatures for up to 3 months without losing efficacy, especially against HPV16. This study is critical, as a thermostable vaccine will decrease vaccine cost associated with cold-chain maintenance and could increase vaccine access and coverage, especially in remote regions of the world.

Immunization with phage virus-like particles displaying Zika virus potential B-cell epitopes neutralizes Zika virus infection of monkey kidney cells.

Zika virus (ZIKV) is a mosquito-borne flavivirus that has re-emerged and is associated with many debilitating clinical manifestations. Research is currently being conducted to develop a prophylactic vaccine against the virus; however, there has not been any licensed ZIKV vaccine. Recent studies have identified potential B-cell epitopes (amino acids 241-259, 294-315, 317-327, 346-361, 377-388 and 421-437) on the envelope protein of ZIKV, which could be explored to develop peptide vaccines against ZIKV infection. Nevertheless, the immunogenicity of these epitopes has never been assessed. Here, we displayed these epitopes on highly immunogenic bacteriophage virus-like particles (VLPs; MS2, PP7 and Qß) platforms and assessed their immunogenicity in mice. Mice immunized with a mixture of VLPs displaying ZIKV envelope B-cell epitopes elicited anti-ZIKV antibodies. Although, immunized mice were not protected against a high challenge dose of ZIKV, sera - albeit at low titers - from immunized mice neutralized (in vitro) a low dose of ZIKV. Taken together, these results show that these epitopes are B-cell epitopes and they are immunogenic when displayed on a Qß VLP platform. Furthermore, the results also show that immunization with VLPs displaying a single B-cell epitope minimally reduces ZIKV infection whereas immunization with a mixture of VLPs displaying a combination of the B-cell epitopes neutralizes ZIKV infection. Thus, immunization with a mixture of VLPs displaying multiple ZIKV B-cell epitopes is a good strategy to enhance ZIKV neutralization.

A novel candidate HPV vaccine: MS2 phage VLP displaying a tandem HPV L2 peptide offers similar protection in mice to Gardasil-9.

Human papillomaviruses (HPVs) cause approximately 5% of cancer cases worldwide. Fortunately, three prophylactic vaccines have been approved to protect against HPV infections. Gardasil-9, the most recent HPV vaccine, is predicted to offer protection against the HPV types that cause ∼90% of cervical cancer, 86% of HPV-associated penile cancers, and ∼93% of HPV-associated head & neck cancers. As an alternative to Gardasil-9, we developed and tested a novel candidate vaccine targeting conserved epitopes in the HPV minor capsid protein, L2. We displayed a tandem HPV31/16L2 peptide (amino acid 17-31) or consensus peptides from HPV L2 (amino acid 69-86 or 108-122) on the surface of bacteriophage MS2 virus-like particles (VLPs). Mice immunized with the MS2 VLPs displaying the tandem peptide or immunized with a mixture of VLPs (displaying the tandem peptide and consensus peptide 69-86) elicited high titer antibodies against individual L2 epitopes. Moreover, vaccinated mice were protected from cervicovaginal infection with HPV pseudoviruses 16, 31, 45, 58 and sera from immunized mice neutralized HPV pseudoviruses 18 and 33 at levels similar to mice immunized with Gardasil-9. These results suggest that immunization with a tandem, L2 peptide or a low valency mixture of L2 peptide-displaying VLPs can provide broad protection against multiple HPV types.

Gardasil-9: A global survey of projected efficacy.

Human papillomaviruses (HPVs) are the causative agents of human neoplasias such as warts and cancers. There are ∼19 HPV types associated with cancers, which has made it very challenging for first generation HPV vaccines to offer complete protection against all cancer-causing HPV types. Recently, a second generation HPV vaccine, Gardasil-9, has been approved to protect against more HPV types. Worldwide, Gardasil-9 will protect against HPV types associated with ∼90% of cervical cancer case in women and 80-95% of other HPV-associated anogenital cancers in both men and women. However, due to variation in HPV-type specific prevalence and distribution, the vaccine will offer different percentages of protection in different geographical regions; Gardasil-9 will offer protection against HPV types associated with ∼87.7% of cervical cancers in Asia, 91.7% in Africa, 92% in North America, 90.9% in Europe, 89.5% in Latin America & the Caribbean, and 86.5% in Australia. Because of this, Pap smear screening and testing for HPV types not included in Gardasil-9 will need to continue, especially in HIV/AIDS patients. In order to achieve complete protection against all HPV types that cause cervical cancer, a third-generation HPV vaccine is needed.

Up-regulation of c-Jun NH2-terminal kinase-interacting protein 3 (JIP3) contributes to BDNF-enhanced neurotransmitter release.

Brain-derived neurotrophic factor (BDNF) has been implicated in the potent modulation of synaptic plasticity at both pre-synaptic and post-synaptic sites. However, the molecular mechanism underlying BDNF-mediated pre-synaptic modulation remains incompletely understood. Here, we report that BDNF treatment for over 4 h could significantly enhance the expression of c-Jun NH2-terminal kinase-interacting protein 3 (JIP3) in cultured hippocampal neurons. This enhancement could be blocked by the Trk inhibitor K252a or by a cAMP response element-binding protein (CREB) inhibitor. In addition, chromatin immunoprecipitation (ChIP) assays revealed that CREB could bind with the JIP3 promoter region and the BDNF treatment could increase this binding. Using dual-luciferase assays we further characterized the cAMP response element (CRE) site in the JIP3 promoter. Finally, we found that BDNF-increased JIP3 expression contributes to the BDNF-induced modulation of neurotransmitter release. Together, our studies reveal that in hippocampal neurons BDNF up-regulates JIP3 expression via CREB activation, which contributes to the enhancement of neurotransmitter release; thus, we have identified a novel mechanism that BDNF modulates pre-synaptic transmission.

c-Jun NH2-terminal kinase (JNK)-interacting protein-3 (JIP3) regulates neuronal axon elongation in a kinesin- and JNK-dependent manner.

The development of neuronal polarity is essential for the establishment of the accurate patterning of neuronal circuits in the brain. However, little is known about the underlying molecular mechanisms that control rapid axon elongation during neuronal development. Here, we report that c-Jun NH2-terminal kinase (JNK)-interacting protein-3 (JIP3) is highly expressed at axon tips during the critical period for axon development. Using gain- and loss-of-function approaches, immunofluorescence analysis, and in utero electroporation, we find that JIP3 can enhance axon elongation in primary hippocampal neurons and cortical neurons in vivo. We further demonstrate that JIP3 promotes axon elongation in a kinesin- and JNK-dependent manner using several deletion mutants of JIP3. Next, we demonstrate that the successful transportation of JIP3 to axon tips by kinesin is a prerequisite for enhancing JNK phosphorylation in this area and therefore promotes axon elongation, constituting a novel mechanism for coupling JIP3 anterograde transport with JNK signaling at the distal axons and axon elongation. Finally, our immunofluorescence data suggest that the activation of JNK at axon tips facilitates axon elongation by modulating cofilin activity and actin filament dynamics. These findings may have important implications for our understanding of neuronal axon elongation during development.