Patient Factors Associated with Missed Otolaryngology Appointments at an Urban Safety-Net Hospital.

OBJECTIVE: To determine if patient factors related to ethnicity, socioeconomic status (SES), medical comorbidities, or appointment characteristics increase the risk of missing an initial adult otolaryngology appointment. METHODS: This study is a retrospective case control study at Boston Medical Center (BMC) in Boston, Massachusetts, that took place in 2019. Patient demographic and medical comorbidity data as well as appointment characteristic data were collected and compared between those that attended their initial otolaryngology appointment versus those who missed their initial appointment. Chi-square and ANOVA tests were used to calculate differences between attendance outcomes. Multivariate analysis was used to compare the odds of missing an appointment based on various patient- and appointment-related factors. RESULTS: Patients who were more likely to miss their appointments were more often female, of lower education, disabled, not employed, Black or Hispanic, and Spanish-speaking. Spring and Fall appointments were more likely to be missed. When a multivariate regression was conducted to control for social determinants of health (SDOH) such as race, insurance status, employment, and education status, the odds of females, Spanish-speaking, students, and disabled patients missing their appointment were no longer statistically significant. CONCLUSION: A majority of patients at BMC come from lower SES backgrounds and have multiple medical comorbidities. Those who reside closer to BMC, often areas of lower average income, had higher rates of missed appointments. Interventions such as decreasing lag time, providing handicap-accessible free transportation, and increasing accessibility of telemedicine for patients could help improve attendance rates at BMC. LEVEL OF EVIDENCE: IV Laryngoscope, 134:4003-4010, 2024.

Detailed mapping of determinants within the porcine endogenous retrovirus envelope surface unit identifies critical residues for human cell infection within the proline-rich region.

Replication-competent porcine endogenous retroviruses (PERVs) are either human cell tropic (PERV-A and PERV-B) or non-human cell tropic (PERV-C). We previously demonstrated that PERV in vitro cell tropism is modulated by 2 residues within the C terminus of SU and that the PERV receptor binding domain (RBD) extends beyond the variable regions A and B (VRA and VRB, respectively), to include the proline rich-region (PRR) of SU (M. Gemeniano et al., Virology 346:108-117, 2000; T. Argaw et al., J. Virol. 82:7483-7489, 2008). The present study aimed to identify the specific elements within the PERV RBD that interact with the C-terminal elements of SU to facilitate human cell infection. We constructed a series of chimeric and mutated envelopes between PERV-A and PERV-C and using pseudotyped retroviral vectors to map the human cell tropism-determining sequences within the PERV RBD. We show that the PRR from PERV-A is both necessary and sufficient to allow human cell infection when substituted into the homologous region of the PERV-C envelope carrying two C-terminal amino acid substitutions shown to influence human cell tropism, Q374R and I412V (PERV-Crv). Furthermore, substitution of a single amino acid residue in the PRR of the non-human-tropic PERV-Crv envelope allows vectors carrying this envelope to infect human cells. Receptor interference assays showed that these modified PERV-C envelopes do not bind either of the human PERV-A receptors, suggesting the presence of a distinct human PERV-C receptor. Finally, vectors carrying these modified PERV-C envelopes infect primary human endothelial cells, a cell type likely to be exposed to PERV in clinical use of certain porcine xenotransplantation products.

Induction of ebolavirus cross-species immunity using retrovirus-like particles bearing the Ebola virus glycoprotein lacking the mucin-like domain.

BACKGROUND: The genus Ebolavirus includes five distinct viruses. Four of these viruses cause hemorrhagic fever in humans. Currently there are no licensed vaccines for any of them; however, several vaccines are under development. Ebola virus envelope glycoprotein (GP1,2) is highly immunogenic, but antibodies frequently arise against its least conserved mucin-like domain (MLD). We hypothesized that immunization with MLD-deleted GP1,2 (GPΔMLD) would induce cross-species immunity by making more conserved regions accessible to the immune system. METHODS: To test this hypothesis, mice were immunized with retrovirus-like particles (retroVLPs) bearing Ebola virus GPΔMLD, DNA plasmids (plasmo-retroVLP) that can produce such retroVLPs in vivo, or plasmo-retroVLP followed by retroVLPs. RESULTS: Cross-species neutralizing antibody and GP1,2-specific cellular immune responses were successfully induced. CONCLUSION: Our findings suggest that GPΔMLD presented through retroVLPs may provide a strategy for development of a vaccine against multiple ebolaviruses. Similar vaccination strategies may be adopted for other viruses whose envelope proteins contain highly variable regions that may mask more conserved domains from the immune system.

Identification of two distinct structural regions in a human porcine endogenous retrovirus receptor, HuPAR2, contributing to function for viral entry.

BACKGROUND: Of the three subclasses of Porcine Endogenous Retrovirus (PERV), PERV-A is able to infect human cells via one of two receptors, HuPAR1 or HuPAR2. Characterizing the structure-function relationships of the two HuPAR receptors in PERV-A binding and entry is important in understanding receptor-mediated gammaretroviral entry and contributes to evaluating the risk of zoonosis in xenotransplantation. RESULTS: Chimeras of the non-permissive murine PAR and the permissive HuPAR2, which scanned the entire molecule, revealed that the first 135 amino acids of HuPAR2 are critical for PERV-A entry. Within this critical region, eighteen single residue differences exist. Site-directed mutagenesis used to map single residues confirmed the previously identified L109 as a binding and infectivity determinant. In addition, we identified seven residues contributing to the efficiency of PERV-A entry without affecting envelope binding, located in multiple predicted structural motifs (intracellular, extracellular and transmembrane). We also show that expression of HuPAR2 in a non-permissive cell line results in an average 11-fold higher infectivity titer for PERV-A compared to equal expression of HuPAR1, although PERV-A envelope binding is similar. Chimeras between HuPAR-1 and -2 revealed that the region spanning amino acids 152-285 is responsible for the increase of HuPAR2. Fine mapping of this region revealed that the increased receptor function required the full sequence rather than one or more specific residues. CONCLUSION: HuPAR2 has two distinct structural regions. In one region, a single residue determines binding; however, in both regions, multiple residues influence receptor function for PERV-A entry.

Identification of residues outside of the receptor binding domain that influence the infectivity and tropism of porcine endogenous retrovirus.

Identification of determinants of human tropism of porcine endogenous retrovirus (PERV) is critical to understanding the risk of transmission of PERV to recipients of porcine xenotransplantation products. Previously, we showed that a chimeric envelope cDNA encoding the 360 N-terminal residues of the human-tropic PERV envelope class A (PERV-A) SU and the 130 C-terminal residues of the pig-tropic PERV-C SU and all of TM (PERV-A/C) showed a 100-fold decrease in infectivity titer on human cells (M. Gemeniano, O. Mpanju, D. R. Salomon, M. V. Eiden, and C. A. Wilson, Virology 346:108-117, 2006). To identify residues important for human cell infection, we performed site-directed mutagenesis on each of the nine residues, singly or in combination, that distinguish the C-terminal region of PERV-C from PERV-A. Of the nine amino acids, two single-amino-acid substitutions, Q374R and I412V, restored the infectivity of human cells to the chimeric PERV-A/C to a titer equivalent to that of PERV-A. In contrast, PERV-A/C mutant envelope Q439P resulted in undetectable infection of human cells and an approximately 1,000-fold decrease in control pig cells. Mutation of K441R rescued mutants that carried Q439P, suggesting an incompatibility between the proline residue at this position and the presence of KK in the proteolytic cleavage signal. We confirmed this incompatibility with vectors carrying PERV-A envelope mutant R462K that were also rendered noninfectious. Finally, tropism of vectors carrying PERV-C envelope mutants with only four amino acid changes in the C terminus of PERV-C envelope, NHRQ436YNRP plus K441R, was shifted to one similar to that of PERV-A. Our results show an important and previously unrecognized role for infectivity and tropism for residues at the C terminus of SU.

The US and EU regulatory perspectives on the clinical use of hematopoietic stem/progenitor cells genetically modified ex vivo by retroviral vectors.

A primary safety issue presented by human hematopoietic stem cells/progenitor cells (HS/PC) genetically modified by gammaretroviral or lentiviral vectors is the risk of oncogenesis. This risk is a potential consequence of either of the following events: (a) the possible unintended generation of replication-competent vector-derived viruses (replication-competent retrovirus, RCR; replication-competent lentivirus, RCL) leading to neoplasia due to RCR/RCL infection of target and nontarget cells in vivo, or (b) intended vector integration in the chromosomal DNA of the target somatic cells leading to neoplasia due to insertional mutagenesis. These risks should be addressed in nonclinical and clinical studies. In the US and the EU, a combination of regulations and guidance documents are available to investigators and sponsors of gene therapy clinical trials. Guidance documents provide a facile way to adapt regulatory recommendations, in line with the changing state of the art in medical science. In the field of retroviral vectors, a number of innovations are being tested in nonclinical or clinical investigations, and each of these will raise their own regulatory issues. Some recent examples of these types of innovations include development of novel vector structures to minimize risks associated with vector integration, such as lentiviral vectors currently used in clinical trials for HS/PC modification that have been designed with deletions of the strong retroviral enhancer associated with oncogenesis.

BCL2L1 is associated with γ-globin gene expression.

Fetal hemoglobin (HbF) expression is partially governed by the trans-acting quantitative trait loci BCL11A and MYB and a cis-acting locus linked to the HBB gene cluster. Our previous analysis of the Genotype-Tissue Expression database suggested that BCL2L1 was associated with HbF gene expression. In erythroid progenitors from patients with sickle cell disease, BCL2L1 messenger RNA (mRNA) levels were positively correlated with HBG mRNA and total HbF concentration (r2 = 0.72, P = .047 and r2 = 0.68, P = .01, respectively). Inhibition of BCL2L1 protein activity in HbF-expressing HUDEP-1 cells decreased HBG expression in a dose-dependent manner. Overexpression of BCL2L1 in these cells increased HBG expression fourfold (P < .05) and increased F cells by 13% (P < .05). Overexpression of BCL2L1 in erythroid progenitors derived from primary adult CD34+ cells upregulated HBG expression 11-fold (P < .05), increased F cells by 18% (P < .01), did not significantly affect cell differentiation or proliferation, and had a minor effect on survival. Although the mechanism remains unknown, our results suggest that BCL2L1 is associated with HbF gene activation.

Advancing Public Health Using Regulatory Science to Enhance Development and Regulation of Medical Products: Food and Drug Administration Research at the Center for Biologics Evaluation and Research.

Center for Biologics Evaluation and Research enhances and supports regulatory decision-making and policy development. This work contributes to our regulatory mission, advances medical product development, and supports Food and Drug Administration's regulatory response to public health crises. This review presents some examples of our diverse scientific work undertaken in recent years to support our regulatory and public health mission.

Three cysteine residues of SLC52A1, a receptor for the porcine endogenous retrovirus-A (PERV-A), play a critical role in cell surface expression and infectivity.

Porcine endogenous retrovirus-A (PERV-A), a gammaretrovirus, infects human cells in vitro, thus raising the potential risk of cross-species transmission in xenotransplantation. Two members of the solute carrier family 52 (SLC52A1 and SLC52A2) are PERV-A receptors. Site-directed mutagenesis of the cDNA encoding SLC52A1 identified that only one of two putative glycosylation signals is occupied by glycans. In addition, we showed that glycosylation of SLC52A1 is not necessary for PERV-A receptor function. We also identified that at a minimum, three cysteine residues are sufficient for SLC52A1 cell surface expression. Mutation of cysteine at position 365 and either of the two cysteine residues in the C-terminal tail at positions 442 or 446 reduced SLC52A1 surface expression and PERV-A infection suggesting that these residues may contribute to overall structural stability and receptor function. Understanding interactions between PERV-A and its cellular receptor may provide novel strategies to prevent zoonotic infection in the setting of xenotransplantation.

Identification of two amino acid residues on Ebola virus glycoprotein 1 critical for cell entry.

Using site-directed mutagenesis and retroviral vector pseudotyping of the wild type or mutated glycoprotein of Zaire ebolavirus (ZEBOV), we analyzed 15 conserved residues in the N-terminus of the filovirus glycoprotein 1 (GP1) in order to identify residues critical for cell entry. Results from infectivity assays and Western blot analyses identified two phenylalanine residues at positions 88 and 159 that appear to be critical for ZEBOV entry in vitro. We extended this observation by introduction of alanines at either position 88 or 159 of Ivory Coast Ebolavirus (CIEBOV) and observed the same phenotype. Further, we showed that introduction of each of the two mutations in a recombinant full-length clone of ZEBOV (Mayinga strain) that also carried the coding sequence for GFP could not be rescued, suggesting the mutants rendered the virus non-infectious. The two phenylalanines that are critical for both ZEBOV and CIEBOV entry are found in two linear domains of GP1 that are highly conserved among filoviruses, and thus could provide a target for rational development of broadly cross-protective vaccines or antiviral therapies.

Mutations altering the gammaretrovirus endoproteolytic motif affect glycosylation of the envelope glycoprotein and early events of the virus life cycle.

Previously, we found that mutation of glutamine to proline in the endoproteolytic cleavage signal of the PERV-C envelope (RQKK to RPKK) resulted in non-infectious vectors. Here, we show that RPKK results in a non-infectious vector when placed in not only a PERV envelope, but also the envelope of a related gammaretrovirus, FeLV-B. The amino acid substitutions do not prevent envelope precursor cleavage, viral core and genome assembly, or receptor binding. Rather, the mutations result in the formation of hyperglycosylated glycoprotein and a reduction in the reverse transcribed minus strand synthesis and undetectable 2-LTR circular DNA in cells exposed to vectors with these mutated envelopes. Our findings suggest novel functions associated with the cleavage signal sequence that may affect trafficking through the glycosylation machinery of the cell. Further, the glycosylation status of the envelope appears to impact post-binding events of the viral life cycle, either membrane fusion, internalization, or reverse transcription.

Development of a real time quantitative PCR assay for detection of porcine endogenous retrovirus.

Real time PCR technology was applied to the development of assays for detection and quantitation of porcine endogenous retrovirus (PERV) RNA and DNA sequences in tissues and cells of human or animal origin. A plasmid construct encoding the PERV-pol gene or the in vitro transcribed RNA derived from the plasmid (cRNA) serves as a standard template for amplification of a 178 bp fragment. This study showed that the detection of this target sequence was linear over a range from 20 copies to 2 million copies of the plasmid and from 100 copies to 1 million copies of the cRNA. In addition, amplification of the target sequence was not inhibited by the presence of exogenous genomic DNA. These results demonstrate that a real time (TaqMan-based) PCR or RT-PCR assay can provide a sensitive, reproducible, and robust method for detecting and quantifying PERV DNA or RNA sequences in samples of human or guinea pig origin.

FDA's Activities Supporting Regulatory Application of "Next Gen" Sequencing Technologies.

Applications of next-generation sequencing (NGS) technologies require availability and access to an information technology (IT) infrastructure and bioinformatics tools for large amounts of data storage and analyses. The U.S. Food and Drug Administration (FDA) anticipates that the use of NGS data to support regulatory submissions will continue to increase as the scientific and clinical communities become more familiar with the technologies and identify more ways to apply these advanced methods to support development and evaluation of new biomedical products. FDA laboratories are conducting research on different NGS platforms and developing the IT infrastructure and bioinformatics tools needed to enable regulatory evaluation of the technologies and the data sponsors will submit. A High-performance Integrated Virtual Environment, or HIVE, has been launched, and development and refinement continues as a collaborative effort between the FDA and George Washington University to provide the tools to support these needs. The use of a highly parallelized environment facilitated by use of distributed cloud storage and computation has resulted in a platform that is both rapid and responsive to changing scientific needs. The FDA plans to further develop in-house capacity in this area, while also supporting engagement by the external community, by sponsoring an open, public workshop to discuss NGS technologies and data formats standardization, and to promote the adoption of interoperability protocols in September 2014. LAY ABSTRACT: Next-generation sequencing (NGS) technologies are enabling breakthroughs in how the biomedical community is developing and evaluating medical products. One example is the potential application of this method to the detection and identification of microbial contaminants in biologic products. In order for the U.S. Food and Drug Administration (FDA) to be able to evaluate the utility of this technology, we need to have the information technology infrastructure and bioinformatics tools to be able to store and analyze large amounts of data. To address this need, we have developed the High-performance Integrated Virtual Environment, or HIVE. HIVE uses a combination of distributed cloud storage and distributed cloud computations to provide a platform that is both rapid and responsive to support the growing and increasingly diverse scientific and regulatory needs of FDA scientists in their evaluation of NGS in research and ultimately for evaluation of NGS data in regulatory submissions.

Comparison of the convergent receptor utilization of a retargeted feline leukemia virus envelope with a naturally-occurring porcine endogenous retrovirus A.

In vitro screening of randomized FeLV Envelope libraries identified the CP isolate, which enters cells through HuPAR-1, one of two human receptors utilized by porcine endogenous retrovirus-A (PERV-A), a distantly related gammaretrovirus. The CP and PERV-A Envs however, share little amino acid homology. Their receptor utilization was examined to define the common receptor usage of these disparate viral Envs. We demonstrate that the receptor usage of CP extends to HuPAR-2 but not to the porcine receptor PoPAR, the cognate receptor for PERV-A. Reciprocal interference between virus expressing CP and PERV-A Envs was observed on human cells. Amino acid residues localized to within the putative second extracellular loop (ECL-2) of PAR-1 and PAR-2 are found to be critical for CP envelope function. Through a panel of receptor chimeras and point mutations, this area was also found to be responsible for the differential usage of the PoPAR receptor between CP and PERV-A.

Development and characterization of rabbit and mouse antibodies against ebolavirus envelope glycoproteins.

Ebolaviruses are the etiologic agents of severe viral hemorrhagic fevers in primates, including humans, and could be misused for the development of biological weapons. The ability to rapidly detect and differentiate these viruses is therefore crucial. Antibodies that can detect reliably the ebolavirus surface envelope glycoprotein GP1,2 or a truncated variant that is secreted from infected cells (sGP) are required for advanced development of diagnostic assays such as sandwich ELISAs or Western blots (WB). We used a GP1,2 peptide conserved among Bundibugyo, Ebola, Reston, Sudan, and Taï Forest viruses and a mucin-like domain-deleted Sudan virus GP1,2 (SudanGPΔMuc) to immunize mice or rabbits, and developed a panel of antibodies that either cross-react or are virus-specific. These antibodies detected full-length GP1,2 and sGP in different assays such as ELISA, FACS, or WB. In addition, some of the antibodies were shown to have potential clinical relevance, as they detected ebolavirus-infected cells by immunofluorescence assay and gave a specific increase in signal by sandwich ELISA against sera from mouse-adapted Ebola virus-infected mice over uninfected mouse sera. Rabbit anti-SudanGPΔMuc polyclonal antibody neutralized gammaretroviral particles pseudotyped with Sudan virus GP1,2, but not particles pseudotyped with other ebolavirusGP1,2. Together, our results suggest that this panel of antibodies may prove useful for both in vitro analyses of ebolavirus GP1,2, as well as analysis of clinically relevant samples.

Phenylalanines at positions 88 and 159 of Ebolavirus envelope glycoprotein differentially impact envelope function.

The envelope glycoprotein (GP) of Ebolavirus (EBOV) mediates viral entry into host cells. Through mutagenesis, we and other groups reported that two phenylalanines at positions 88 and 159 of GP are critical for viral entry. However, it remains elusive which steps of viral entry are impaired by F88 or F159 mutations and how. In this study, we further characterized these two phenylalanines through mutagenesis and examined the impact on GP expression, function, and structure. Our data suggest that F159 plays an indirect role in viral entry by maintaining EBOV GP's overall structure. In contrast, we did not detect any evidence for conformational differences in GP with F88 mutations. The data suggest that F88 influences viral entry during a step after cathepsin processing, presumably impacting viral fusion.

Functional hierarchy of two L domains in porcine endogenous retrovirus (PERV) that influence release and infectivity.

The porcine endogenous retrovirus (PERV) Gag protein contains two late (L) domain motifs, PPPY and P(F/S)AP. Using viral release assays we demonstrate that PPPY is the dominant L domain involved in PERV release. PFAP represents a novel retroviral L domain variant and is defined by abnormal viral assembly phenotypes visualized by electron microscopy and attenuation of early PERV release as measured by viral genomes. PSAP is functionally dominant over PFAP in early PERV release. PSAP virions are 3.5-fold more infectious in vitro by TCID(50) and in vivo results in more RNA positive tissues and higher levels of proviral DNA using our human PERV-A receptor (HuPAR-2) transgenic mouse model [Martina, Y., Marcucci, K.T., Cherqui, S., Szabo, A., Drysdale, T., Srinivisan, U., Wilson, C.A., Patience, C., Salomon, D.R., 2006. Mice transgenic for a human porcine endogenous retrovirus receptor are susceptible to productive viral infection. J. Virol. 80 (7), 3135-3146]. The functional hierarchies displayed by PERV L domains, demonstrates that L domain selection in viral evolution exists to promote efficient viral assembly, release and infectivity in the virus-host context.

No evidence of PERV infection in healthcare workers exposed to transgenic porcine liver extracorporeal support.

BACKGROUND: Clinical xenotransplantation holds great promise by providing one solution to the shortage of human organs for transplantation, while also posing a potential public health threat by facilitating transmission of infectious disease from source animals to humans. One potential vector for infectious disease transmission is healthcare workers (HCW) who are involved in administering xenotransplantation procedures. METHODS: In this study, we studied 49 healthcare workers involved in the care of two subjects who participated in a study of porcine liver perfusion as treatment of fulminant hepatic failure. We looked for serologic and virologic evidence of transmission of porcine endogenous retrovirus, and found that HCW had no evidence of infection. CONCLUSIONS: Results of our survey demonstrate that application of standard precautions may be sufficient to prevent transmission of porcine endogenous retrovirus, an agent of concern in ex vivo xenotransplantation products.

The infectivity and host range of the ecotropic porcine endogenous retrovirus, PERV-C, is modulated by residues in the C-terminal region of its surface envelope protein.

Endogenous retroviral genetic material serves as a reservoir for the generation of retroviral pathogens by recombination between activated endogenous or exogenous infectious agents. Some porcine tissues actively express infectious porcine endogenous retroviruses (PERVs). Of the three classes of PERV characterized to date, two, PERV-A and B, are capable of infecting human cells in vitro, whereas PERV-C cannot. Here, we demonstrate that the PERV-C envelope surface protein (SU) when disassociated from its C-terminus binds human cells. Further, we show that PERV-C binding to human cells is not inhibited in 293 cells productively infected with PERV-A, confirming that the molecule PERV-C interacts with on human cells is distinct from that used by PERV-A. Moreover, we demonstrate that the envelope region encompassing the proline-rich region is required for binding to cells in addition to the putative variable region A (VRA) and B (VRB). The region in the C-terminus of the SU that alters the binding and infectivity properties of PERV-C differs by only nine residues from the analogous region of PERV-A. Caution may be warranted even when a xenotransplantation product is from source pigs that do not express human-tropic viruses, as minimal mutations within PERV-C combined with selection in a human recipient could render PERV-C infectious in humans.

Limited infection without evidence of replication by porcine endogenous retrovirus in guinea pigs.

Porcine endogenous retrovirus (PERV) may potentially be transmitted through porcine xenotransplantation products administered to humans. This study examined the feasibility of using guinea pigs as a model to characterize the in vivo infectivity of PERV. To enhance the susceptibility of guinea pigs to retroviral infection or genomic integration, moderate physiological or immunological changes were induced prior to exposing the animals to PERV. Quantitative PERV-specific PCR performed on all tested samples resulted in either undetectable or very low copy numbers of proviruses, even in animals possessing PERV-specific antibody responses. The low copy number of viral DNA detected suggests that PERV infected a limited number of cells. However, PERV DNA levels did not increase over time, suggesting no virus replication occurred. These results in the guinea pig are similar to previous observations of non-human primate cells that allow PERV infection but do not support PERV replication in vitro.