Structure-based design of a soluble human cytomegalovirus glycoprotein B antigen stabilized in a prefusion-like conformation.

Human cytomegalovirus (HCMV) glycoprotein B (gB) is a class III membrane fusion protein required for viral entry. HCMV vaccine candidates containing gB have demonstrated moderate clinical efficacy, but no HCMV vaccine has been approved. Here, we used structure-based design to identify and characterize amino acid substitutions that stabilize gB in its metastable prefusion conformation. One variant containing two engineered interprotomer disulfide bonds and two cavity-filling substitutions (gB-C7), displayed increased expression and thermostability. A 2.8 Å resolution cryoelectron microscopy structure shows that gB-C7 adopts a prefusion-like conformation, revealing additional structural elements at the membrane-distal apex. Unlike previous observations for several class I viral fusion proteins, mice immunized with postfusion or prefusion-stabilized forms of soluble gB protein displayed similar neutralizing antibody titers, here specifically against an HCMV laboratory strain on fibroblasts. Collectively, these results identify initial strategies to stabilize class III viral fusion proteins and provide tools to probe gB-directed antibody responses.

A single, improbable B cell receptor mutation confers potent neutralization against cytomegalovirus.

Cytomegalovirus (CMV) is a leading cause of infant hearing loss and neurodevelopmental delay, but there are no clinically licensed vaccines to prevent infection, in part due to challenges eliciting neutralizing antibodies. One of the most well-studied targets for CMV vaccines is the viral fusogen glycoprotein B (gB), which is required for viral entry into host cells. Within gB, antigenic domain 2 site 1 (AD-2S1) is a target of potently neutralizing antibodies, but gB-based candidate vaccines have yet to elicit robust responses against this region. We mapped the genealogy of B cells encoding potently neutralizing anti-gB AD-2S1 antibodies from their inferred unmutated common ancestor (UCA) and characterized the binding and function of early lineage ancestors. Surprisingly, we found that a single amino acid heavy chain mutation A33N, which was an improbable mutation rarely generated by somatic hypermutation machinery, conferred broad CMV neutralization to the non-neutralizing UCA antibody. Structural studies revealed that this mutation mediated key contacts with the gB AD-2S1 epitope. Collectively, these results provide insight into potently neutralizing gB-directed antibody evolution in a single donor and lay a foundation for using this B cell-lineage directed approach for the design of next-generation CMV vaccines.

Human Cytomegalovirus mRNA-1647 Vaccine Candidate Elicits Potent and Broad Neutralization and Higher Antibody-Dependent Cellular Cytotoxicity Responses Than the gB/MF59 Vaccine.

BACKGROUND: MF59-adjuvanted gB subunit (gB/MF59) vaccine demonstrated approximately 50% efficacy against human cytomegalovirus (HCMV) acquisition in multiple clinical trials, suggesting that efforts to improve this vaccine design might yield a vaccine suitable for licensure. METHODS: A messenger RNA (mRNA)-based vaccine candidate encoding HCMV gB and pentameric complex (PC), mRNA-1647, is currently in late-stage efficacy trials. However, its immunogenicity has not been compared to the partially effective gB/MF59 vaccine. We assessed neutralizing and Fc-mediated immunoglobulin G (IgG) effector antibody responses induced by mRNA-1647 in both HCMV-seropositive and -seronegative vaccinees from a first-in-human clinical trial through 1 year following third vaccination using a systems serology approach. Furthermore, we compared peak anti-gB antibody responses in seronegative mRNA-1647 vaccinees to that of seronegative gB/MF59 vaccine recipients. RESULTS: mRNA-1647 vaccination elicited and boosted HCMV-specific IgG responses in seronegative and seropositive vaccinees, respectively, including neutralizing and Fc-mediated effector antibody responses. gB-specific IgG responses were lower than PC-specific IgG responses. gB-specific IgG and antibody-dependent cellular phagocytosis responses were lower than those elicited by gB/MF59. However, mRNA-1647 elicited higher neutralization and antibody-dependent cellular cytotoxicity (ADCC) responses. CONCLUSIONS: Overall, mRNA-1647 vaccination induced polyfunctional and durable HCMV-specific antibody responses, with lower gB-specific IgG responses but higher neutralization and ADCC responses compared to the gB/MF59 vaccine. CLINICAL TRIALS REGISTRATION: NCT03382405 (mRNA-1647) and NCT00133497 (gB/MF59).

PPP2R2A prostate cancer haploinsufficiency is associated with worse prognosis and a high vulnerability to B55α/PP2A reconstitution that triggers centrosome destabilization.

The PPP2R2A gene encodes the B55α regulatory subunit of PP2A. Here, we report that PPP2R2A is hemizygously lost in ~42% of prostate adenocarcinomas, correlating with reduced expression, poorer prognosis, and an increased incidence of hemizygous loss (>75%) in metastatic disease. Of note, PPP2R2A homozygous loss is less common (5%) and not increased at later tumor stages. Reduced expression of B55α is also seen in prostate tumor tissue and cell lines. Consistent with the possibility that complete loss of PPP2R2A is detrimental in prostate tumors, PPP2R2A deletion in cells with reduced but present B55α reduces cell proliferation by slowing progression through the cell cycle. Remarkably, B55α-low cells also appear addicted to lower B55α expression, as even moderate increases in B55α expression are toxic. Reconstitution of B55α expression in prostate cancer (PCa) cell lines with low B55α expression reduces proliferation, inhibits transformation and blocks xenograft tumorigenicity. Mechanistically, we show B55α reconstitution reduces phosphorylation of proteins essential for centrosomal maintenance, and induces centrosome collapse and chromosome segregation failure; a first reported link between B55α/PP2A and the vertebrate centrosome. These effects are dependent on a prolonged metaphase/anaphase checkpoint and are lethal to PCa cells addicted to low levels of B55α. Thus, we propose the reduction in B55α levels associated with hemizygous loss is necessary for centrosomal integrity in PCa cells, leading to selective lethality of B55α reconstitution. Such a vulnerability could be targeted therapeutically in the large pool of patients with hemizygous PPP2R2A deletions, using pharmacologic approaches that enhance PP2A/B55α activity.

The changing role of incentives in health promotion and wellness.

Incentives have been a part of the health promotion and wellness landscape for over three decades. The mounting need for engagement and risk mitigation are changing the role that incentives play in programming strategies. Incentives are increasing in size and are undergoing a fast process of assimilation within large employer organizations. More sophisticated communication strategies are helping maximize the motive force element in many settings. Health plans have been slower to innovate with incentives but are now attempting to catch up. A possible "ideal" or "endgame" incentive strategy is suggested and one scenario for the future of wellness incentives is proposed.

Characterizing the point spread function of retinal OCT devices with a model eye-based phantom.

We have designed, fabricated, and tested a nanoparticle-embedded phantom (NEP) incorporated into a model eye in order to characterize the point spread function (PSF) of retinal optical coherence tomography (OCT) devices in three dimensions under realistic imaging conditions. The NEP comprises a sparse distribution of highly backscattering silica-gold nanoshells embedded in a transparent UV-curing epoxy. The commercially-available model eye replicates the key optical structures and focusing power of the human eye. We imaged the model eye-NEP combination with a research-grade spectral domain OCT system designed for in vivo retinal imaging and quantified the lateral and axial PSF dimensions across the field of view in the OCT images. We also imaged the model eye-NEP in a clinical OCT system. Subtle features in the PSF and its dimensions were consistent with independent measurements of lateral and axial resolution. This model eye-based phantom can provide retinal OCT device developers and users a means to rapidly, objectively, and consistently assess the PSF, a fundamental imaging performance metric.

Optical coherence tomography imaging of retinal damage in real time under a stimulus electrode.

We have developed a novel method to study the effects of electrical stimulation of the local retina directly under an epiretinal stimulus electrode in real time. Using optical coherence tomography (OCT) and a superfused retinal eyecup preparation, we obtained high-resolution images of the rabbit retina directly under an optically transparent saline-filled fluoropolymer stimulation tube electrode. During OCT imaging, 50 Hz trains of biphasic current pulses 1 ms/phase (23-749 µC cm(-2) ph(-1)) were applied to the retinal surface for 5 min. After imaging, the stimulated regions were stained with the dye propidium iodide (PI) to reveal cytotoxic damage. Pulse train stimulation at 44-133 µC cm(-2) ph(-1) had little effect on the retina; however, trains ≥442 µC cm(-2) ph(-1) caused increases in the reflectance of the inner plexiform layer (IPL) and edema. The damage seen in retinal OCT images matched the pattern observed in histological sections, and in the PI staining. With pulse trains ≥442 µC cm(-2) ph(-1), rapid increases in the reflectivity of the IPL could be observed under the stimulus electrode. Below the electrode, we observed a ring-like pattern of retinal detachment in the subretinal space. The OCT imaging method may be useful for analyzing overstimulation of neuronal tissue by electrodes in many brain regions.