Parainfluenza virus 5 (PIV5) amplifying virus-like particles expressing respiratory syncytial virus (RSV) antigens protect mice against RSV infection.

Respiratory syncytial virus (RSV) is the leading cause of bronchiolitis and pneumonia in children under one year of age. In addition to causing severe respiratory diseases in children, it is also a major cause of morbidity and mortality among the elderly and immunocompromised individuals. RSV is the most common cause of lower respiratory tract infections, yet there are currently no licensed vaccines. A parainfluenza virus 5 (PIV5)-based amplifying virus-like particle (AVLP), which enables the use of PIV5 RNA transcription/replication machinery to express gene of interest, has recently been developed. We evaluated the PIV5-based AVLP system as a vaccine platform for RSV by incorporating the fusion protein (F) gene and the transcription factor protein (M2-1) gene of RSV into the PIV5-AVLP backbone (AVLP-F and AVLP-M2-1, respectively). Mice immunized with a single dose of the AVLP-F or AVLP-M2-1 developed RSV-F or RSV-M2-1-specific immune responses, respectively. Both vaccine candidates elicited antigen-specific cell-mediated responses at levels comparable to or higher than an RSV infection. Most importantly, each vaccine was able to induce protection against RSV A2 challenge in the mouse model. These results indicate the potential of the PIV5-based AVLP system as a platform for vaccines against RSV infection.

Developing a platform system for gene delivery: amplifying virus-like particles (AVLP) as an influenza vaccine.

Delivery of a gene of interest to target cells is highly desirable for translational medicine, such as gene therapy, regenerative medicine, vaccine development, and studies of gene function. Parainfluenza virus 5 (PIV5), a paramyxovirus with a negative-sense RNA genome, normally infects cells without causing obvious cytopathic effect, and it can infect many cell types. To exploit these features of PIV5, we established a system generating self-amplifying, virus-like particles (AVLP). Using enhanced green fluorescent protein (EGFP) as a reporter, AVLP encoding EGFP (AVLP-EGFP) successfully delivered and expressed the EGFP gene in primary human cells, including stem cells, airway epithelial cells, monocytes, and T cells. To demonstrate the application of this system for vaccine development, we generated AVLPs to express the HA and M1 antigens from the influenza A virus strain H5N1 (AVLP-H5 and AVLP-M1H5). Immunization of mice with AVLP-H5 and AVLP-M1H5 generated robust antibody and cellular immune responses. Vaccination with a single dose of AVLP-H5 and M1H5 completely protected mice against lethal H5N1 challenge, suggesting that the AVLP-based system is a promising platform for delivery of desirable genes.

Establishing a small animal model for evaluating protective immunity against mumps virus.

Although mumps vaccines have been used for several decades, protective immune correlates have not been defined. Recently, mumps outbreaks have occurred in vaccinated populations. To better understand the causes of the outbreaks and to develop means to control outbreaks in mumps vaccine immunized populations, defining protective immune correlates will be critical. Unfortunately, no small animal model for assessing mumps immunity exists. In this study, we evaluated use of type I interferon (IFN) alpha/beta receptor knockout mice (IFN-α/ßR-/-) for such a model. We found these mice to be susceptible to mumps virus administered intranasally and intracranially. Passive transfer of purified IgG from immunized mice protected naïve mice from mumps virus infection, confirming the role of antibody in protection and demonstrating the potential for this model to evaluate mumps immunity.

Oligomerization of Mumps Virus Phosphoprotein.

UNLABELLED: The mumps virus (MuV) genome encodes a phosphoprotein (P) that is important for viral RNA synthesis. P forms the viral RNA-dependent RNA polymerase with the large protein (L). P also interacts with the viral nucleoprotein (NP) and self-associates to form a homotetramer. The P protein consists of three domains, the N-terminal domain (P(N)), the oligomerization domain (P(O)), and the C-terminal domain (P(C)). While P(N) is known to relax the NP-bound RNA genome, the roles of P(O) and P(C) are not clear. In this study, we investigated the roles of P(O) and P(C) in viral RNA synthesis using mutational analysis and a minigenome system. We found that P(N) and P(C) functions can be trans-complemented. However, this complementation requires P(O), indicating that P(O) is essential for P function. Using this trans-complementation system, we found that P forms parallel dimers (P(N) to P(N) and P(C) to P(C)). Furthermore, we found that residues R231, K238, K253, and K260 in P(O) are critical for P's functions. We identified P(C) to be the domain that interacts with L. These results provide structure-function insights into the role of MuV P. IMPORTANCE: MuV, a paramyxovirus, is an important human pathogen. The P protein of MuV is critical for viral RNA synthesis. In this work, we established a novel minigenome system that allows the domains of P to be complemented in trans. Using this system, we confirmed that MuV P forms parallel dimers. An understanding of viral RNA synthesis will allow the design of better vaccines and the development of antivirals.

Evaluation of pre-radiotherapy apparent diffusion coefficient (ADC): patterns of recurrence and survival outcomes analysis in patients treated for glioblastoma multiforme.

PURPOSE: To investigate the association of pre-radiotherapy apparent diffusion coefficient (ADC) abnormalities with patterns of recurrence and outcomes in patients with glioblastoma multiforme (GBM). MATERIALS AND METHODS: Fifty-two patients with recurrent GBM were retrospectively evaluated. Diffusion MRI images were acquired for all patients postoperatively prior to radiotherapy. ADC images were evaluated for geographic regions of diffusion restriction (hypointensity) within the FLAIR volume. If identified, the ADC map and the T1+C MRI at the time of recurrence were registered to the original plan to determine the pattern of recurrence and the coverage of the ADC abnormality by the 60 Gy isodose line (IDL). Progression-free and overall survival was determined for patients with and without an ADC hypointensity. RESULTS: An ADC hypointensity was identified in 32 (62%) of cases. The recurrence pattern in these cases was central in 27/32 (84%), marginal in 4/32 (13%) and distant in 1/32 (3%). The recurrence overlapped with the ADC hypointensity in 28 (88%) patients. The ADC hypointensity was covered by 95% of the 60 Gy IDL in all cases. Kaplan-Meier analysis revealed inferior progression free survival and overall survival in patients with an ADC hypointensity compared to those without, despite similarities between the groups in terms of age, RT dose, performance status, and extent of resection. CONCLUSIONS: The presence of an ADC hypointensity on pre-radiotherapy diffusion-weighted imaging is associated with the location of tumor recurrence as demonstrated by frequent overlap in this series, and is associated with a trend toward inferior outcomes. This abnormality may reflect a high risk region of hypercellularity and warrants consideration with respect to radiotherapy planning.

Evaluation of absolute and normalized apparent diffusion coefficient (ADC) values within the post-operative T2/FLAIR volume as adverse prognostic indicators in glioblastoma.

To evaluate the association of normalized and absolute ADC metrics with progression free survival (PFS) and overall survival (OS) in patients treated for glioblastoma multiforme (GBM). Fifty-two patients with preradiotherapy diffusion weighted imaging treated with post-operative chemoradiation for GBM were evaluated. Region of interest analysis for ADC metrics including mean and minimum ADC value (ADCmean) and (ADCmin) was performed within the T2/FLAIR volume. Normalized (N)ADC values were generated relative to contralateral white matter. PFS and OS were analyzed relative to ADC parameters using a regression model. Kaplan-Meier and Cox proportional hazards analysis with respect to (N)ADCmean, and (N)ADCmin was performed. A (N)ADC threshold <1.3 within the T2/FLAIR volume was analyzed with respect to PFS and OS. Regression analysis indicated that normalized ADC values provide the strongest association with PFS and OS. Kaplan-Meier analysis revealed a non-significant trend toward inferior PFS and OS associated with (N)ADCmean <1.7, and a significant decrement to PFS and OS associated with (N)ADCmin <0.3. (N)ADCmin was a significant prognostic factor when taking into account age, performance status, and extent of resection. ADC thresholding analysis revealed that a retained volume of >0.45 cc per mL FLAIR volume was associated with a trend toward inferior PFS and OS. In the post-operative, pre-radiotherapy setting, the (N)ADCmin is the strongest predictor of outcomes in patients treated for GBM. ADC thresholding analysis indicates that a large volume of normalized ADC value <1.3 may be associated with adverse outcomes.

Use of Helical TomoTherapy for the Focal Hypofractionated Treatment of Limited Brain Metastases in the Initial and Recurrent Setting.

BACKGROUND: Whole-brain radiation therapy (WBRT), stereotactic radiosurgery (SRS), or both are commonly employed in the treatment of limited brain metastases in the initial or recurrent setting. Hypofractionated partial volume irradiation is also employed, however, published experience using helical TomoTherapy (HT) for this purposes is limited. We reviewed our institutional experience to assess patient selection factors, fractionation scheme, and outcomes associated with this technique. METHODS: A retrospective chart review was performed to evaluate patients treated with partial volume hypofractionated HT-based IMRT for brain metastases at our institution. RESULTS: Thirteen patients (7M/6F, median age 62, median KPS 90) with a limited (1-9) number of brain metastases in the primary or recurrent setting were identified. Primary malignancies included colorectal (3), NSCLC (5), RCC (1), breast (1), melanoma (1), uterine (1), and ovarian (1). The median time from initial diagnosis to brain metastases was 20.7 months (range 0-61.3). Treatment was delivered to intact metastases in six patients, to a single resection cavity in six patients, and to both in one patient. A total of 27 lesions were treated. The median number of intact metastases treated was two (range 1-9). Previous treatments included WBRT (5), WBRT + SRS (3), SRS alone (1), and none (4). The most common fractionation schemes were 25 Gy in five fractions and 27.5 Gy in five fractions to each lesion. At a median of 6 months follow up (range 1.26-20.13) after TomoTherapy, 10 patients were deceased, 2 were alive, and 1 was lost to follow up. Systemic progression occurred in seven patients and intracranial progression occurred in five. The median intracranial progression free survival and overall survival after TomoTherapy was 6.3 months. Freedom from local failure for treated lesions was 71% and 59% at 6 and 12 months. CONCLUSION: TomoTherapy-based hypofractionated radiotherapy to a limited number of metastatic lesions is associated with acceptable intracranial disease control and survival outcomes and represents a viable treatment option in the primary and recurrent setting for select patients.

Structural studies on the authentic mumps virus nucleocapsid showing uncoiling by the phosphoprotein.

Mumps virus (MuV) is a highly contagious pathogen, and despite extensive vaccination campaigns, outbreaks continue to occur worldwide. The virus has a negative-sense, single-stranded RNA genome that is encapsidated by the nucleocapsid protein (N) to form the nucleocapsid (NC). NC serves as the template for both transcription and replication. In this paper we solved an 18-Å-resolution structure of the authentic MuV NC using cryo-electron microscopy. We also observed the effects of phosphoprotein (P) binding on the MuV NC structure. The N-terminal domain of P (PNTD) has been shown to bind NC and appeared to induce uncoiling of the helical NC. Additionally, we solved a 25-Å-resolution structure of the authentic MuV NC bound with the C-terminal domain of P (PCTD). The location of the encapsidated viral genomic RNA was defined by modeling crystal structures of homologous negative strand RNA virus Ns in NC. Both the N-terminal and C-terminal domains of MuV P bind NC to participate in access to the genomic RNA by the viral RNA-dependent-RNA polymerase. These results provide critical insights on the structure-function of the MuV NC and the structural alterations that occur through its interactions with P.

Effect of treatment modality on the hypothalamic-pituitary function of patients treated with radiation therapy for pituitary adenomas: hypothalamic dose and endocrine outcomes.

BACKGROUND: Both fractionated external beam radiotherapy and single fraction radiosurgery for pituitary adenomas are associated with the risk of hypothalamic-pituitary (HP) axis dysfunction. OBJECTIVE: To analyze the effect of treatment modality (Linac, TomoTherapy, or gamma knife) on hypothalamic dose and correlate these with HP-axis deficits after radiotherapy. METHODS: Radiation plans of patients treated post-operatively for pituitary adenomas using Linac-based 3D-conformal radiotherapy (CRT) (n = 11), TomoTherapy-based intensity modulated radiation therapy (IMRT) (n = 10), or gamma knife stereotactic radiosurgery (n = 12) were retrospectively reviewed. Dose to the hypothalamus was analyzed and post-radiotherapy hormone function including growth hormone, thyroid stimulating hormone, adrenocorticotropic hormone, prolactin, and gonadotropins (follicle stimulating hormone/luteinizing hormone) were assessed. RESULTS: Post-radiation, 13 of 27 (48%) patients eligible for analysis developed at least one new hormone deficit, of which 8 of 11 (72%) occurred in the Linac group, 4 of 8 (50%) occurred in the TomoTherapy group, and 1 of 8 (12.5%) occurred in the gamma knife group. Compared with fractionated techniques, gamma knife showed improved hypothalamic sparing for DMax Hypo and V12Gy. For fractionated modalities, TomoTherapy showed improved dosimetric characteristics over Linac-based treatment with hypothalamic DMean (44.8 vs. 26.8 Gy p = 0.02), DMax (49.8 vs. 39.1 Gy p = 0.04), and V12Gy (100 vs. 76% p = 0.004). CONCLUSION: Maximal dosimetric avoidance of the hypothalamus was achieved using gamma knife-based radiosurgery followed by TomoTherapy-based IMRT, and Linac-based 3D conformal radiation therapy, respectively.

Roles of serine and threonine residues of mumps virus P protein in viral transcription and replication.

UNLABELLED: Mumps virus (MuV), a paramyxovirus containing a negative-sense nonsegmented RNA genome, is a human pathogen that causes an acute infection with symptoms ranging from parotitis to mild meningitis and severe encephalitis. Vaccination against mumps virus has been effective in reducing mumps cases. However, recently large outbreaks have occurred in vaccinated populations. There is no anti-MuV drug. Understanding replication of MuV may lead to novel antiviral strategies. MuV RNA-dependent RNA polymerase minimally consists of the phosphoprotein (P) and the large protein (L). The P protein is heavily phosphorylated. To investigate the roles of serine (S) and threonine (T) residues of P in viral RNA transcription and replication, P was subjected to mass spectrometry and mutational analysis. P, a 392-amino acid residue protein, has 64 S and T residues. We have found that mutating nine S/T residues significantly reduced and mutating residue T at 101 to A (T101A) significantly enhanced activity in a minigenome system. A recombinant virus containing the P-T101A mutation (rMuV-P-T101A) was recovered and analyzed. rMuV-P-T101A grew to higher titers and had increased protein expression at early time points. Together, these results suggest that phosphorylation of MuV-P-T101 plays a negative role in viral RNA synthesis. This is the first time that the P protein of a paramyxovirus has been systematically analyzed for S/T residues that are critical for viral RNA synthesis. IMPORTANCE: Mumps virus (MuV) is a reemerging paramyxovirus that caused large outbreaks in the United States, where vaccination coverage is very high. There is no anti-MuV drug. In this work, we have systematically analyzed roles of Ser/Thr residues of MuV P in viral RNA synthesis. We have identified S/T residues of P critical for MuV RNA synthesis and phosphorylation sites that are important for viral RNA synthesis. This work leads to a better understanding of viral RNA synthesis as well as to potential novel strategies to control mumps.

Substrate recognition reduces side-chain flexibility for conserved hydrophobic residues in human Pin1.

Pin1 is a peptidyl-prolyl isomerase consisting of a WW domain and a catalytic isomerase (PPIase) domain connected by a flexible linker. Pin1 recognizes phospho-Ser/Thr-Pro motifs in cell-signaling proteins, and is both a cancer and an Alzheimer's disease target. Here, we provide novel insight into the functional motions underlying Pin1 substrate interaction using nuclear magnetic resonance deuterium ((2)D) and carbon ((13)C) spin relaxation. Specifically, we compare Pin1 side-chain motions in the presence and absence of a known phosphopeptide substrate derived from the mitotic phosphatase Cdc25. Substrate interaction alters Pin1 side-chain motions on both the microsecond-millisecond (mus-ms) and picosecond-nanosecond (ps-ns) timescales. Alterations include loss of ps-ns flexibility along an internal conduit of hydrophobic residues connecting the catalytic site with the interdomain interface. These residues are conserved among Pin1 homologs; hence, their dynamics are likely important for the Pin1 mechanism.

p38 MAP kinase activity is correlated with angiotensin II type 1 receptor blocker-induced left ventricular reverse remodeling in spontaneously hypertensive heart failure rats.

BACKGROUND: Angiotensin II type 1 receptor blocker L-158,809 (ARB) induces reverse left ventricular (LV) remodeling in spontaneously hypertensive heart failure (SHHF) rats. However, the signaling mechanism that mediates ARB-induced reverse LV remodeling remains unclear. The present study was to determine if changes in mitogen-activated protein kinase (MAPK, including ERK, JNK, and p38) signaling correlate with ARB-elicited reversal of cardiac hypertrophy in SHHF rats. METHODS AND RESULTS: In 1 set of experiments, 5-month-old lean female SHHF rats were treated with L-158,809 (ARB) or the vasodilator hydralazine (HYD) for 1 month, respectively. In a second set of experiments, 5-month-old SHHF rats were treated with ARB for 6 months or 1 month and then with HYD for 5 months. Either ARB or HYD normalized left ventricular end systolic pressure in SHHF rats relative to normotensive control Wistar Furth (WF) rats at both 6 and 11 months of age, but only ARB reduced heart-to-body weight ratio in SHHF rats to control level. Western blot analysis showed that cardiac p38 MAPK activity was markedly increased in 6-month-old SHHF rats, but dramatically reduced in 11-month-old SHHF rats compared with WF rats, as indicated by the levels of phosphorylated form of p38. The alterations in p38 activity were completely reversed by ARB treatment but not by HYD treatment. CONCLUSION: ARB restored normal cardiac p38 activity, which coincided with ARB-induced reverse LV remodeling in SHHF rats, suggesting a strong correlation between p38 signaling and cardiac remodeling.