RG2-VLP: a Vaccine Designed to Broadly Protect against Anogenital and Skin Human Papillomaviruses Causing Human Cancer.

The family of human papillomaviruses (HPV) includes over 400 genotypes. Genus α genotypes generally infect the anogenital mucosa, and a subset of these HPV are a necessary, but not sufficient, cause of cervical cancer. Of the 13 high-risk (HR) and 11 intermediate-risk (IR) HPV associated with cervical cancer, genotypes 16 and 18 cause 50% and 20% of cases, respectively, whereas HPV16 dominates in other anogenital and oropharyngeal cancers. A plethora of ßHPVs are associated with cutaneous squamous cell carcinoma (CSCC), especially in sun-exposed skin sites of epidermodysplasia verruciformis (EV), AIDS, and immunosuppressed patients. Licensed L1 virus-like particle (VLP) vaccines, such as Gardasil 9, target a subset of αHPV but no ßHPV. To comprehensively target both α- and ßHPVs, we developed a two-component VLP vaccine, RG2-VLP, in which L2 protective epitopes derived from a conserved αHPV epitope (amino acids 17 to 36 of HPV16 L2) and a consensus ßHPV sequence in the same region are displayed within the DE loop of HPV16 and HPV18 L1 VLP, respectively. Unlike vaccination with Gardasil 9, vaccination of wild-type and EV model mice (Tmc6Δ/Δ or Tmc8Δ/Δ) with RG2-VLP induced robust L2-specific antibody titers and protected against ß-type HPV5. RG2-VLP protected rabbits against 17 αHPV, including those not covered by Gardasil 9. HPV16- and HPV18-specific neutralizing antibody responses were similar between RG2-VLP- and Gardasil 9-vaccinated animals. However, only transfer of RG2-VLP antiserum effectively protected naive mice from challenge with all ßHPVs tested. Taken together, these observations suggest RG2-VLP's potential as a broad-spectrum vaccine to prevent αHPV-driven anogenital, oropharyngeal, and ßHPV-associated cutaneous cancers. IMPORTANCE Licensed preventive HPV vaccines are composed of VLPs derived by expression of major capsid protein L1. They confer protection generally restricted to infection by the αHPVs targeted by the up-to-9-valent vaccine, and their associated anogenital cancers and genital warts, but do not target ßHPV that are associated with CSCC in EV and immunocompromised patients. We describe the development of a two-antigen vaccine protective in animal models against known oncogenic αHPVs as well as diverse ßHPVs by incorporation into HPV16 and HPV18 L1 VLP of 20-amino-acid conserved protective epitopes derived from minor capsid protein L2.

Passive Immunization with a Single Monoclonal Neutralizing Antibody Protects against Cutaneous and Mucosal Mouse Papillomavirus Infections.

We have established a mouse papillomavirus (MmuPV1) model that induces both cutaneous and mucosal infections and cancers. In the current study, we use this model to test our hypothesis that passive immunization using a single neutralizing monoclonal antibody can protect both cutaneous and mucosal sites at different time points after viral inoculation. We conducted a series of experiments involving the administration of either a neutralizing monoclonal antibody, MPV.A4, or control monoclonal antibodies to both outbred and inbred athymic mice. Three clinically relevant mucosal sites (lower genital tract for females and anus and tongue for both males and females) and two cutaneous sites (muzzle and tail) were tested. At the termination of the experiments, all tested tissues were harvested for virological analyses. Significantly lower levels of viral signals were detected in the MPV.A4-treated female mice up to 6 h post-viral inoculation compared to those in the isotype control. Interestingly, males displayed partial protection when they received MPV.A4 at the time of viral inoculation, even though they were completely protected when receiving MPV.A4 at 24 h before viral inoculation. We detected MPV.A4 in the blood starting at 1 h and up to 8 weeks postadministration in some mice. Parallel to these in vivo studies, we conducted in vitro neutralization using a mouse keratinocyte cell line and observed complete neutralization up to 8 h post-viral inoculation. Thus, passive immunization with a monoclonal neutralizing antibody can protect against papillomavirus infection at both cutaneous and mucosal sites and is time dependent. IMPORTANCE This is the first study testing a single monoclonal neutralizing antibody (MPV.A4) by passive immunization against papillomavirus infections at both cutaneous and mucosal sites in the same host in the mouse papillomavirus model. We demonstrated that MPV.A4 administered before viral inoculation can protect both male and female athymic mice against MmuPV1 infections at cutaneous and mucosal sites. MPV.A4 also offers partial protection at 6 h post-viral inoculation in female mice. MPV.A4 can be detected in the blood from 1 h to 8 weeks after intraperitoneal (i.p.) injection. Interestingly, males were only partially protected when they received MPV.A4 at the time of viral inoculation. The failed protection in males was due to the absence of neutralizing MPV.A4 at the infected sites. Our findings suggest passive immunization with a single monoclonal neutralizing antibody can protect against diverse papillomavirus infections in a time-dependent manner in mice.

Helical TomoTherapy Total Lymphoid Irradiation and Hematopoietic Cell Transplantation for Kidney Transplant Tolerance in Rhesus Macaques.

Development of a post-transplant kidney transplant tolerance induction protocol involving a novel total lymphoid irradiation (TLI) conditioning method in a rhesus macaque model is described. We examined the feasibility of acheiving tolerance to MHC 1-haplotype matched kidney transplants by establishing a mixed chimeric state with infusion of donor hematopoietic cells (HC) using TomoTherapy TLI. The chimeric state was hypothesized to permit the elimination of all immunosuppressive (IS) medications while preserving allograft function long-term without development of graft-versus-host-disease (GVHD) or rejection. An experimental group of 11 renal transplant recipients received the tolerance induction protocol and outcomes were compared to a control group (n = 7) that received the same conditioning but without donor HC infusion. Development of mixed chimerism and operational tolerance was accomplished in two recipients in the experimental group. Both recipients were withdrawn from all IS and continued to maintain normal renal allograft function for 4 years without rejection or GVHD. None of the animals in the control group achieved tolerance when IS was eliminated. This novel experimental model demonstrated the feasibility for inducing of long-term operational tolerance when mixed chimerism is achieved using a TLI post-transplant conditioning protocol in 1-haplotype matched non-human primate recipients of combined kidney and HC transplantation.

Adaptation-proof SARS-CoV-2 vaccine design.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) surface spike glycoprotein - a major antibody target - is critical for virus entry via engagement of human angiotensin-converting enzyme 2 (ACE2) receptor. Despite successes with existing vaccines and therapies that primarily target the receptor binding domain (RBD) of the spike protein, the susceptibility of RBD to mutations provides escape routes for the SARS-CoV-2 from neutralizing antibodies. On the other hand, structural conservation in the spike protein can be targeted to reduce escape mutations and achieve broad protection. Here, we designed candidate stable immunogens that mimic surface features of selected conserved regions of spike protein through 'epitope grafting,' in which we present the target epitope topology on diverse heterologous scaffolds that can structurally accommodate the spike epitopes. Structural characterization of the epitope-scaffolds showed stark agreement with our computational models and target epitopes. The sera from mice immunized with engineered designs display epitope-scaffolds and spike binding activity. We also demonstrated the utility of the designed epitope-scaffolds in diagnostic applications. Taken all together, our study provides important methodology for targeting the conserved, non-RBD structural motifs of spike protein for SARS-CoV-2 epitope vaccine design and demonstrates the potential utility of 'epitope grafting' in rational vaccine design.

Comprehensive Assessment of the Antigenic Impact of Human Papillomavirus Lineage Variation on Recognition by Neutralizing Monoclonal Antibodies Raised against Lineage A Major Capsid Proteins of Vaccine-Related Genotypes.

Human papillomavirus (HPV) is the causative agent of cervical and other epithelial cancers. Naturally occurring variants of HPV have been classified into lineages and sublineages based on their whole-genome sequences, but little is known about the impact of this diversity on the structure and function of viral gene products. The HPV capsid is an icosahedral lattice comprising 72 pentamers of the major capsid protein (L1) and the associated minor capsid protein (L2). We investigated the potential impact of this genome variation on the capsid antigenicity of lineage and sublineage variants of seven vaccine-relevant, oncogenic HPV genotypes by using a large panel of monoclonal antibodies (MAbs) raised against the L1 proteins of lineage A antigens. Each genotype had at least one variant that displayed a ≥4-fold reduced neutralizing antibody sensitivity against at least one MAb, demonstrating that naturally occurring variation can affect one or more functional antigenic determinants on the HPV capsid. For HPV16, HPV18, HPV31, and HPV45, the overall impact was of a low magnitude. For HPV33 (sublineages A2 and A3 and lineages B and C), HPV52 (lineage D), and HPV58 (lineage C), however, variant residues in the indicated lineages and sublineages reduced their sensitivity to neutralization by all MAbs by up to 1,000-fold, suggesting the presence of key antigenic determinants on the surface of these capsids. These determinants were resolved further by site-directed mutagenesis. These data improve our understanding of the impact of naturally occurring variation on the antigenicity of the HPV capsid of vaccine-relevant oncogenic HPV genotypes.IMPORTANCE Human papillomavirus (HPV) is the causative agent of cervical and some other epithelial cancers. HPV vaccines generate functional (neutralizing) antibodies that target the virus particles (or capsids) of the most common HPV cancer-causing genotypes. Each genotype comprises variant forms that have arisen over millennia and which include changes within the capsid proteins. In this study, we explored the potential for these naturally occurring variant capsids to impact recognition by neutralizing monoclonal antibodies. All genotypes included at least one variant form that exhibited reduced recognition by at least one antibody, with some genotypes affected more than others. These data highlight the impact of naturally occurring variation on the structure of the HPV capsid proteins of vaccine-relevant oncogenic HPV genotypes.

Definitive-intent intensity modulated radiotherapy for modified-Adams' stage 4 canine sinonasal cancer: A retrospective study of 29 cases (2011-2017).

Dogs with sinonasal tumors with cribriform plate lysis (modified Adams' stage 4) treated with non-conformal definitive radiotherapy (RT) have short median survivals of 6-7 months. Intensity-modulated radiotherapy with its greater conformality and tumor dose homogeneity may result in more favorable outcomes. Dogs with epithelial or mesenchymal sinonasal tumors and CT evidence of cribriform lysis that received 10 daily fractions of 4.2 Gray using IMRT by helical tomotherapy were included in this single-institution retrospective case series study. Dogs with distant metastasis, previous treatment, or concurrent chemotherapy were excluded. Based on CT, tumors were divided into two groups: cribriform plate lysis only (stage 4a) or intracranial extension (stage 4b). Twenty-nine dogs were included, 23 with carcinoma and six with sarcoma. Eight dogs had stage 4b tumors; two presented with neurologic signs. Two dogs had lymph node metastasis at diagnosis, one confirmed and one suspected. Radiation dose distributions were standardized and patient positioning for RT was verified daily using on-board megavoltage CT. All evaluable dogs had improvement of clinical signs. Median progression free survival was 177 days (95% CI, 128-294 days). Median overall survival was 319 days (95% CI, 188-499 days). Radiotherapy was well tolerated. The most common side effect was grade 1 or 2 oral mucositis. Two dogs that received additional treatment at progression (stereotactic RT [1]; surgery [1]) developed significant late effects. Image-guided definitive-intent IMRT may improve survival in dogs with modified Adams' stage 4 sinonasal tumors and is associated with low morbidity. Intracranial tumor extension was not prognostic in this cohort of uniformly treated dogs.

Definitive-intent intensity-modulated radiation therapy provides similar outcomes to those previously published for definitive-intent three-dimensional conformal radiation therapy in dogs with primary brain tumors: A multi-institutional retrospective study.

Radiotherapy with or without surgery is a common choice for brain tumors in dogs. Although numerous studies have evaluated use of three-dimensional conformal radiotherapy, reports of definitive-intent, IMRT for canine intracranial tumors are lacking. Intensity-modulated radiation therapy has the benefit of decreasing dose to nearby organs at risk and may aid in reducing toxicity. However, increasing dose conformity with IMRT calls for accurate target delineation and daily patient positioning, in order to decrease the risk of a geographic miss. To determine survival outcome and toxicity, we performed a multi-institutional retrospective observational study evaluating dogs with brain tumors treated with IMRT. Fifty-two dogs treated with fractionated, definitive-intent IMRT at four academic radiotherapy facilities were included. All dogs presented with neurologic signs and were diagnosed via MRI. Presumed radiological diagnoses included 37 meningiomas, 12 gliomas, and one peripheral nerve sheath tumor. One dog had two presumed meningiomas and one dog had either a glioma or meningioma. All dogs were treated in the macroscopic disease setting and were prescribed a total dose of 45-50 Gy (2.25-2.5 Gy per fraction in 18-20 daily fractions). Median survival time for all patients, including seven cases treated with a second course of therapy was 18.1 months (95% confidence of interval 12.3-26.6 months). As previously described for brain tumors, increasing severity of neurologic signs at diagnosis was associated with a worse outcome. Intensity-modulated radiation therapy was well tolerated with few reported acute, acute delayed, or late side effects.

Short survival time following palliative-intent hypofractionated radiotherapy for non-resectable canine thyroid carcinoma: A retrospective analysis of 20 dogs.

Radiotherapy is the treatment of choice for non-resectable canine thyroid carcinoma. High tumor response rates and median survival times of 2 years or longer have been previously reported with conventionally fractionated and hypofractionated protocols, even in dogs with distant metastasis. The objective of this retrospective, descriptive, case series study was to evaluate the clinical outcomes of dogs with thyroid carcinoma irradiated with palliative intent using hypofractionated radiotherapy at our institution. Medical records of 20 dogs treated between 1999 and 2014 were reviewed. All dogs had macroscopic primary tumors and presented with tumor-related clinical signs. Median longest tumor diameter was 10 cm. Nineteen dogs (95%) had metastasis (7/19 lymph node; 16/19 distant metastasis). Most dogs were treated with four weekly fractions of 6.5-8 Gy. Radiotherapy was well tolerated in 17 dogs; three died of respiratory compromise before completing radiotherapy. Eleven dogs received adjuvant chemotherapy. Five dogs experienced a local tumor response including two complete and three partial responses. Overall median survival time was 170 days (range, 1-824 days; 95% CI: 58-392 days). Of potential variables examined (radiation delivery system and protocol, tumor size and location, vascular/lymphatic invasion, metastatic disease, chemotherapy, tumor response), only achievement of complete or partial response was predictive of overall survival. In contrast to previously reported cohorts, dogs with clinical signs and stage IV disease predominated in this study. Previous studies may over-estimate survival following hypofractionated radiotherapy in dogs with advanced thyroid carcinoma.

A survey of stereotactic radiation therapy in veterinary medicine.

Radiotherapy plays an important role in curative and palliative cancer treatment. As a novel radiation delivery technique, stereotactic radiotherapy utilizes three-dimensional-conformal treatment planning, high-precision beam delivery technology, and patient specific position verification to target tumors, often in one to five high-dose fractions. Currently, there is no consensus about best stereotactic radiotherapy practices in veterinary radiotherapy. The objective of this study was to document the breadth of perspectives, techniques, and applications of stereotactic radiotherapy in veterinary medicine. We conducted an online survey of American College of Veterinary Radiology members specializing in radiation oncology to assess how, when, and why stereotactic radiotherapy is being used. Both stereotactic radiotherapy users and nonusers completed the survey. The overall response and survey completion rates were 54% (67/123) and 87% (58/67), respectively. Overall, 55% of respondents reported providing stereotactic radiotherapy at their facility, with a median of 4.5 canine cases and one feline case per month. Delivery methods included C-arm linear accelerator with multi-leaf collimator, helical tomotherapy, and CyberKnife. Nonpituitary intracranial tumors, pituitary tumors, and sinonasal tumors were the most common cancers treated using stereotactic radiotherapy in both species. The most common fractionation scheme was three fractions of 10 Gy/fraction. The results of this survey suggest common availability of stereotactic radiotherapy in veterinary radiation facilities. These results provide valuable information regarding current stereotactic radiotherapy practices in veterinary medicine, and may provide an initial step toward standardizing methods and establishing consensus guidelines.

Mouse papillomavirus infections spread to cutaneous sites with progression to malignancy.

We report secondary cutaneous infections in the mouse papillomavirus (MmuPV1)/mouse model. Our previous study demonstrated that cutaneous MmuPV1 infection could spread to mucosal sites. Recently, we observed that mucosal infections could also spread to various cutaneous sites including the back, tail, muzzle and mammary tissues. The secondary site lesions were positive for viral DNA, viral capsid protein and viral particles as determined by in situ hybridization, immunohistochemistry and transmission electron microscopy analyses, respectively. We also demonstrated differential viral production and tumour growth at different secondarily infected skin sites. For example, fewer viral particles were detected in the least susceptible back tissues when compared with those in the infected muzzle and tail, although similar amounts of viral DNA were detected. Follow-up studies demonstrated that significantly lower amounts of viral DNA were packaged in the back lesions. Lavages harvested from the oral cavity and lower genital tracts were equally infectious at both cutaneous and mucosal sites, supporting the broad tissue tropism of this papillomavirus. Importantly, two secondary skin lesions on the forearms of two mice displayed a malignant phenotype at about 9.5 months post-primary infection. Therefore, MmuPV1 induces not only dysplasia at mucosal sites such as the vagina, anus and oral cavity but also skin carcinoma at cutaneous sites. These findings demonstrate that MmuPV1 mucosal infection can be spread to cutaneous sites and suggest that the model could serve a useful role in the study of the viral life cycle and pathogenesis of papillomavirus.

Broad Cross-Protection Is Induced in Preclinical Models by a Human Papillomavirus Vaccine Composed of L1/L2 Chimeric Virus-Like Particles.

UNLABELLED: At least 15 high-risk human papillomaviruses (HPVs) are linked to anogenital preneoplastic lesions and cancer. Currently, there are three licensed prophylactic HPV vaccines based on virus-like particles (VLPs) of the L1 major capsid protein from HPV-2, -4, or -9, including the AS04-adjuvanted HPV-16/18 L1 vaccine. The L2 minor capsid protein contains HPV-neutralizing epitopes that are well conserved across numerous high-risk HPVs. Therefore, the objective of our study was to assess the capacity to broaden vaccine-mediated protection using AS04-adjuvanted vaccines based on VLP chimeras of L1 with one or two L2 epitopes. Several chimeric VLPs were constructed by inserting L2 epitopes within the DE loop and/or C terminus of L1. Based on the shape, yield, size, and immunogenicity, one of seven chimeras was selected for further evaluation in mouse and rabbit challenge models. The chimeric VLP consisted of HPV-18 L1 with insertions of HPV-33 L2 (amino acid residues 17 to 36; L1 DE loop) and HPV-58 L2 (amino acid residues 56 to 75; L1 C terminus). This chimeric L1/L2 VLP vaccine induced persistent immune responses and protected against all of the different HPVs evaluated (HPV-6, -11, -16, -31, -35, -39, -45, -58, and -59 as pseudovirions or quasivirions) in both mouse and rabbit challenge models. The degree and breadth of protection in the rabbit were further enhanced when the chimeric L1/L2 VLP was formulated with the L1 VLPs from the HPV-16/18 L1 vaccine. Therefore, the novel HPV-18 L1/L2 chimeric VLP (alone or in combination with HPV-16 and HPV-18 L1 VLPs) formulated with AS04 has the potential to provide broad protective efficacy in human subjects. IMPORTANCE: From evaluations in human papillomavirus (HPV) protection models in rabbits and mice, our study has identified a prophylactic vaccine with the potential to target a wide range of HPVs linked to anogenital cancer. The three currently licensed vaccines contain virus-like particles (VLPs) of the L1 major capsid protein from two, four, or nine different HPVs. Rather than increasing the diversity of L1 VLPs, this vaccine contains VLPs based on a recombinant chimera of two highly conserved neutralizing epitopes from the L2 capsid protein inserted into L1. Our study demonstrated that the chimeric L1/L2 VLP is an effective vehicle for displaying two different L2 epitopes and can be used in a quantity equivalent to what is used in the licensed vaccines. Hence, using the chimeric L1/L2 VLP may be a more cost-effective approach for vaccine formulation than adding different VLPs for each HPV.

Efficient replication of Epstein-Barr virus in stratified epithelium in vitro.

Epstein-Barr virus is a ubiquitous human herpesvirus associated with epithelial and lymphoid tumors. EBV is transmitted between human hosts in saliva and must cross the oral mucosal epithelium before infecting B lymphocytes, where it establishes a life-long infection. The latter process is well understood because it can be studied in vitro, but our knowledge of infection of epithelial cells has been limited by the inability to infect epithelial cells readily in vitro or to generate cell lines from EBV-infected epithelial tumors. Because epithelium exists as a stratified tissue in vivo, organotypic cultures may serve as a better model of EBV in epithelium than monolayer cultures. Here, we demonstrate that EBV is able to infect organotypic cultures of epithelial cells to establish a predominantly productive infection in the suprabasal layers of stratified epithelium, similar to that seen with Kaposi's-associated herpesvirus. These cells did express latency-associated proteins in addition to productive-cycle proteins, but a population of cells that exclusively expressed latency-associated viral proteins could not be detected; however, an inability to infect the basal layer would be unlike other herpesviruses examined in organotypic cultures. Furthermore, infection did not induce cellular proliferation, as it does in B cells, but instead resulted in cytopathic effects more commonly associated with productive viral replication. These data suggest that infection of epithelial cells is an integral part of viral spread, which typically does not result in the immortalization or enhanced growth of infected epithelial cells but rather in efficient production of virus.

Meningeal dissemination of a pituitary carcinoma to the cauda equina in a dog.

An 8-year-old spayed female border collie dog was diagnosed with an invasive pituitary macrotumor. Five months after radiation therapy, the patient developed paraparesis and lumbosacral pain. Necropsy revealed a pituitary carcinoma with cauda equina drop metastasis. In cases of pituitary masses, meningeal dissemination should be considered if neurologic status declines.

Dissémination méningée d'un carcinome pituitaire à la queue de cheval chez un chien. Une chienne Border collie stérilisée âgée de 8 ans a été diagnostiquée avec une macrotumeur pituitaire invasive. Cinq mois après la radiothérapie, la patiente a développé de la paraparésie et de la douleur lombo-sacrée. La nécropsie a révélé un carcinome pituitaire avec une métastase de la partie inférieure de la queue de cheval. Dans les cas des masses pituitaires, la dissémination méningée devrait être considérée s'il se produit un déclin de l'état neurologique.(Traduit par Isabelle Vallières).

The U4 Antibody Epitope on Human Papillomavirus 16 Identified by Cryo-electron Microscopy.

UNLABELLED: The human papillomavirus (HPV) major structural protein L1 composes capsomers that are linked together through interactions mediated by the L1 C terminus to constitute a T=7 icosahedral capsid. H16.U4 is a type-specific monoclonal antibody recognizing a conformation-dependent neutralizing epitope of HPV thought to include the L1 protein C terminus. The structure of human papillomavirus 16 (HPV16) complexed with H16.U4 fragments of antibody (Fab) was solved by cryo-electron microscopy (cryo-EM) image reconstruction. Atomic structures of virus and Fab were fitted into the corresponding cryo-EM densities to identify the antigenic epitope. The antibody footprint mapped predominately to the L1 C-terminal arm with an additional contact point on the side of the capsomer. This footprint describes an epitope that is presented capsid-wide. However, although the H16.U4 epitope suggests the presence of 360 potential binding sites exposed in the capsid valley between each capsomer, H16.U4 Fab bound only to epitopes located around the icosahedral five-fold vertex of the capsid. Thus, the binding characteristics of H16.U4 defined in this study showed a distinctive selectivity for local conformation-dependent interactions with specific L1 invading arms between five-fold related capsomers. IMPORTANCE: Human papillomavirus 16 (HPV16) is the most prevalent oncogenic genotype in HPV-associated anogenital and oral cancers. Here we use cryo-EM reconstruction techniques to solve the structures of the HPV16 capsid complexes using H16.U4 fragment of antibody (Fab). Different from most other antibodies directed against surface loops, H16.U4 monoclonal antibody is unique in targeting the C-terminal arm of the L1 protein. This monoclonal antibody (MAb) is used throughout the HPV research community in HPV serological and vaccine development and to define mechanisms of HPV uptake. The unique binding mode of H16.U4 defined here shows important conformation-dependent interactions within the HPV16 capsid. By targeting an important structural and conformational epitope, H16.U4 may identify subtle conformational changes in different maturation stages of the HPV capsid and provide a key probe to analyze the mechanisms of HPV uptake during the early stages of virus infection. Our analyses precisely define important conformational epitopes on HPV16 capsids that are key targets for successful HPV prophylactic vaccines.

A cryo-electron microscopy study identifies the complete H16.V5 epitope and reveals global conformational changes initiated by binding of the neutralizing antibody fragment.

UNLABELLED: Human papillomavirus 16 (HPV16) is a worldwide health threat and an etiologic agent of cervical cancer. To understand the antigenic properties of HPV16, we pursued a structural study to elucidate HPV capsids and antibody interactions. The cryo-electron microscopy (cryo-EM) structures of a mature HPV16 particle and an altered capsid particle were solved individually and as complexes with fragment of antibody (Fab) from the neutralizing antibody H16.V5. Fitted crystal structures provided a pseudoatomic model of the virus-Fab complex, which identified a precise footprint of H16.V5, including previously unrecognized residues. The altered-capsid-Fab complex map showed that binding of the Fab induced significant conformational changes that were not seen in the altered-capsid structure alone. These changes included more ordered surface loops, consolidated so-called "invading-arm" structures, and tighter intercapsomeric connections at the capsid floor. The H16.V5 Fab preferentially bound hexavalent capsomers likely with a stabilizing effect that directly correlated with the number of bound Fabs. Additional cryo-EM reconstructions of the virus-Fab complex for different incubation times and structural analysis provide a model for a hyperstabilization of the capsomer by H16.V5 Fab and showed that the Fab distinguishes subtle differences between antigenic sites. IMPORTANCE: Our analysis of the cryo-EM reconstructions of the HPV16 capsids and virus-Fab complexes has identified the entire HPV.V5 conformational epitope and demonstrated a detailed neutralization mechanism of this clinically important monoclonal antibody against HPV16. The Fab bound and ordered the apical loops of HPV16. This conformational change was transmitted to the lower region of the capsomer, resulting in enhanced intercapsomeric interactions evidenced by the more ordered capsid floor and "invading-arm" structures. This study advances the understanding of the neutralization mechanism used by H16.V5.

SINGLE INSTITUTION VARIABILITY IN INTENSITY MODULATED RADIATION TARGET DELINEATION FOR CANINE NASAL NEOPLASIA.

Contouring variability is a significant barrier to the accurate delivery and reporting of radiation therapy. The aim of this descriptive study was to determine the variation in contouring radiation targets and organs at risk by participants within our institution. Further, we also aimed to determine if all individuals contoured the same normal tissues. Two canine nasal tumor datasets were selected and contoured by two ACVR-certified radiation oncologists and two radiation oncology residents from the same institution. Eight structures were consistently contoured including the right and left eye, the right and left lens, brain, the gross tumor volume (GTV), clinical target volume (CTV), and planning target volume (PTV). Spinal cord, hard and soft palate, and bulla were contoured on 50% of datasets. Variation in contouring occurred in both targets and normal tissues at risk and was particularly significant for the GTV, CTV, and PTV. The mean metric score and dice similarity coefficient were below the threshold criteria in 37.5-50% and 12.5-50% of structures, respectively, quantitatively indicating contouring variation. This study refutes our hypothesis that minimal variation in target and normal tissue delineation occurs. The variation in contouring may contribute to different tumor response and toxicity for any given patient. Our results also highlight the difficulty associated with replication of published radiation protocols or treatments, as even with replete contouring description the outcome of treatment is still fundamentally influenced by the individual contouring the patient.

Tracking vaginal, anal and oral infection in a mouse papillomavirus infection model.

Noninvasive and practical techniques to longitudinally track viral infection are sought after in clinical practice. We report a proof-of-principle study to monitor the viral DNA copy number using a newly established mouse papillomavirus (MmuPV1) mucosal infection model. We hypothesized that viral presence could be identified and quantified by collecting lavage samples from cervicovaginal, anal and oral sites. Nude mice infected at these sites with infectious MmuPV1 were tracked for up to 23 weeks starting at 6 weeks post-infection. Viral DNA copy number was determined by SYBR Green Q-PCR analysis. In addition, we tracked viral DNA load through three complete oestrous cycles to pinpoint whether there was a correlation between the DNA load and the four stages of the oestrous cycle. Our results showed that high viral DNA copy number was reproducibly detected from both anal and cervicovaginal lavage samples. The infection and disease progression were further confirmed by histology, cytology, in situ hybridization, immunohistochemistry and transmission electron microscopy. Interestingly, the viral copy number fluctuated over the oestrous cycle, with the highest level at the oestrus stage, implying that multiple sampling might be necessary to provide a reliable diagnosis. Virus DNA was detected in oral lavage samples at a later time after infection. Lower viral DNA load was found in oral samples when compared with those in anal and vaginal tracts. To our knowledge, our study is the first in vivo study to sequentially monitor papillomavirus infection from mucosal anal, oral and vaginal tracts in a preclinical model.

The type 1 diabetes resistance locus B10 Idd9.3 mediates impaired B-cell lymphopoiesis and implicates microRNA-34a in diabetes protection.

NOD.B10 Idd9.3 mice are congenic for the insulin-dependent diabetes (Idd) Idd9.3 locus, which confers significant type 1 diabetes (T1D) protection and encodes 19 genes, including microRNA (miR)-34a, from T1D-resistant C57BL/10 mice. B cells have been shown to play a critical role in the priming of autoantigen-specific CD4(+) T cells in T1D pathogenesis in non-obese diabetic (NOD) mice. We show that early B-cell development is impaired in NOD.B10 Idd9.3 mice, resulting in the profound reduction of transitional and mature splenic B cells as compared with NOD mice. Molecular analysis revealed that miR-34a expression was significantly higher in B-cell progenitors and marginal zone B cells from NOD.B10 Idd9.3 mice than in NOD mice. Furthermore, miR-34a expression in these cell populations inversely correlated with levels of Foxp1, an essential regulator of B-cell lymphopoiesis, which is directly repressed by miR-34a. In addition, we show that islet-specific CD4(+) T cells proliferated inefficiently when primed by NOD.B10 Idd9.3 B cells in vitro or in response to endogenous autoantigen in NOD.B10 Idd9.3 mice. Thus, Idd9.3-encoded miR-34a is a likely candidate in negatively regulating B-cell lymphopoiesis, which may contribute to inefficient expansion of islet-specific CD4(+) T cells and to T1D protection in NOD.B10 Idd9.3 mice.

Protection from tumor recurrence following adoptive immunotherapy varies with host conditioning regimen despite initial regression of autochthonous murine brain tumors.

Adoptive T cell transfer (ACT) has achieved clinical success in treating established cancer, particularly in combination with lymphodepleting regimens. Our group previously demonstrated that ACT following whole-body irradiation (WBI) promotes high-level T cell accumulation, regression of established brain tumors, and long-term protection from tumor recurrence in a mouse model of SV40 T antigen-induced choroid plexus tumors. Here we asked whether an approach that can promote strong donor T-cell responses in the absence of WBI might also produce this dramatic and durable tumor elimination following ACT. Agonist anti-CD40 antibody can enhance antigen-specific CD8(+) T-cell responses and has shown clinical efficacy as a monotherapy in the setting of cancer. We show that anti-CD40 conditioning promotes rapid accumulation of tumor-specific donor CD8(+) T cells in the brain and regression of autochthonous T antigen-induced choroid plexus tumors, similar to WBI. Despite a significant increase in the lifespan, tumors eventually recurred in anti-CD40-conditioned mice coincident with loss of T-cell persistence from both the brain and lymphoid organs. Depletion of CD8(+) T cells from the peripheral lymphoid organs of WBI-conditioned recipients failed to promote tumor recurrence, but donor cells persisted in the brains long-term in CD8-depleted mice. These results demonstrate that anti-CD40 conditioning effectively enhances ACT-mediated acute elimination of autochthonous tumors, but suggest that mechanisms associated with WBI conditioning, such as the induction of long-lived T cells, may be critical for protection from tumor recurrence.

Characterization of a 0.35T MR system for phantom image quality stability and in vivo assessment of motion quantification.

ViewRay is a novel MR-guided radiotherapy system capable of imaging in near real-time at four frames per second during treatment using 0.35T field strength. It allows for improved gating techniques and adaptive radiotherapy. Three cobalt-60 sources (~ 15,000 Curies) permit multiple-beam, intensity-modulated radiation therapy. The primary aim of this study is to assess the imaging stability, accuracy, and automatic segmentation algorithm capability to track motion in simulated and in vivo targets. Magnetic resonance imaging (MRI) characteristics of the system were assessed using the American College of Radiology (ACR)-recommended phantom and accreditation protocol. Images of the ACR phantom were acquired using a head coil following the ACR scanning instructions. ACR recommended T1- and T2-weighted sequences were evaluated. Nine measurements were performed over a period of seven months, on just over a monthly basis, to establish consistency. A silicon dielectric gel target was attached to the motor via a rod. 40 mm total amplitude was used with cycles of 3 to 9 s in length in a sinusoidal trajectory. Trajectories of six moving clinical targets in four canine patients were quantified and tracked. ACR phantom images were analyzed, and the results were compared with the ACR acceptance levels. Measured slice thickness accuracies were within the acceptance limits. In the 0.35 T system, the image intensity uniformity was also within the ACR acceptance limit. Over the range of cycle lengths, representing a wide range of breathing rates in patients imaged at four frames/s, excellent agreement was observed between the expected and measured target trajectories. In vivo canine targets, including the gross target volume (GTV), as well as other abdominal soft tissue structures, were visualized with inherent MR contrast, allowing for preliminary results of target tracking.