Jacqueline A. Hobbs

Next generation of adeno-associated virus 2 vectors: Point mutations in tyrosines lead to high-efficiency transduction at lower doses

Recombinant adeno-associated virus 2 (AAV2) vectors are in use in several Phase I/II clinical trials, but relatively large vector doses are needed to achieve therapeutic benefits. Large vector doses also trigger an immune response as a significant fraction of the vectors fails to traffic efficiently to the nucleus and is targeted for degradation by the host cell proteasome machinery. We have reported that epidermal growth factor receptor protein tyrosine kinase (EGFR-PTK) signaling negatively affects transduction by AAV2 vectors by impairing nuclear transport of the vectors. We have also observed that EGFR-PTK can phosphorylate AAV2 capsids at tyrosine residues. Tyrosine-phosphorylated AAV2 vectors enter cells efficiently but fail to transduce effectively, in part because of ubiquitination of AAV capsids followed by proteasome-mediated degradation. We reasoned that mutations of the surface-exposed tyrosine residues might allow the vectors to evade phosphorylation and subsequent ubiquitination and, thus, prevent proteasome-mediated degradation. Here, we document that site-directed mutagenesis of surface-exposed tyrosine residues leads to production of vectors that transduce HeLa cells ≈10-fold more efficiently in vitro and murine hepatocytes nearly 30-fold more efficiently in vivo at a log lower vector dose. Therapeutic levels of human Factor IX (F.IX) are also produced at an ≈10-fold reduced vector dose. The increased transduction efficiency of tyrosine-mutant vectors is due to lack of capsid ubiquitination and improved intracellular trafficking to the nucleus. These studies have led to the development of AAV vectors that are capable of high-efficiency transduction at lower doses, which has important implications in their use in human gene therapy.

Tyrosine phosphorylation of AAV2 vectors and its consequences on viral intracellular trafficking and transgene expression

We have documented that epidermal growth factor receptor protein tyrosine kinase (EGFR-PTK) signaling negatively affects intracellular trafficking and transduction efficiency of recombinant adeno-associated virus 2 (AAV2) vectors. Specifically, inhibition of EGFR-PTK signaling leads to decreased ubiquitination of AAV2 capsid proteins, which in turn, facilitates viral nuclear transport by limiting proteasome-mediated degradation of AAV2 vectors. In the present studies, we observed that AAV capsids can indeed be phosphorylated at tyrosine residues by EGFR-PTK in in vitro phosphorylation assays and that phosphorylated AAV capsids retain their structural integrity. However, although phosphorylated AAV vectors enter cells as efficiently as their unphosphorylated counterparts, their transduction efficiency is significantly reduced. This reduction is not due to impaired viral second-strand DNA synthesis since transduction efficiency of both single-stranded AAV (ssAAV) and self-complementary AAV (scAAV) vectors is decreased by approximately 68% and approximately 74%, respectively. We also observed that intracellular trafficking of tyrosine-phosphorylated AAV vectors from cytoplasm to nucleus is significantly decreased, which results from ubiquitination of AAV capsids followed by proteasome-mediated degradation, although downstream consequences of capsid ubiquitination may also be affected by tyrosine-phosphorylation. These studies provide new insights into the role of tyrosine-phosphorylation of AAV capsids in various steps in the virus life cycle, which has implications in the optimal use of recombinant AAV vectors in human gene therapy.

Detection of adeno-associated virus 2 and parvovirus b19 in the human dorsolateral prefrontal cortex

Although animal parvoviruses have long been recognized as causes of brain pathology in multiple animal models, especially during early development, human parvoviruses are rarely thought of as neurotropic or causes of neuropathology in humans. However, several recent case reports have suggested possible associations of parvovirus B19 (B19) infection with various neurological and neuropsychiatric symptoms. Adeno-associated virus 2 (AAV2) is related to B19 but has thus far not been shown to be associated with any human disease but is of clinical interest because of the recent use of recombinant AAV vectors in human gene therapy, including gene delivery to the brain. To date, there have been no large-scale studies of the propensity of wild-type human parvoviruses to infect the brain. The Stanley Medical Research Institute Brain Collection offered a unique opportunity to study a large sample (n = 104) of dorsolateral prefrontal cortex (DLPC) DNAs isolated from unaffected control, schizophrenic, and bipolar disorder brains for the presence of parvoviral sequences. This is the first investigator-blinded study to document the presence of parvoviral sequences in the DLPC by utilizing highly sensitive nested polymerase chain reaction (nPCR) and DNA sequencing. Of the overall sample, 6.7% to 12.5% were positive for AAV2, and 14.4% to 42.3% were positive for B19 sequences, with no statistical differences among subgroups. This is the first report to demonstrate the presence of human parvoviruses in a large cohort of adult DLPC, which underscores the need to gain a better insight into the basic biology of parvovirus-brain interactions, including mechanisms of infection and persistence.

Persistent parvovirus B19 infection in non-erythroid tissues: Possible role in the inflammatory and disease process

Parvovirus B19 (B19V) is a small non-enveloped DNA virus of the Parvoviridae family. It is an obligate human pathogen that preferentially replicates in erythroid progenitor cells. B19V is the causative agent of multiple erythroid-related diseases due to replication-induced cytotoxicity. Despite its strong erythroid tropism and related acute disease association, B19V has been determined to persist in many other non-erythroid tissues. This review summarizes and appraises what is known about concomitant B19V DNA persistence and non-acute viral gene expression in various, particularly non-erythroid, tissue types. The methods utilized for B19V detection are described, focusing on the discrepancies in outcomes among the employed assays. The studies where investigations focused on the impact of persistent B19V expression on cellular signaling pathways are also summarized. These studies demonstrate the expanse of the types of cells capable of in vivo B19V expression as well as the possible effect of persistent viral infection on the cellular microenvironment. Overall, these reports indicate that B19V commonly persists in a wide range of both erythroid and non-erythroid tissues, and that low-level viral gene expression can be detected in some persistently infected cells. B19V capsid RNA or proteins have been reported in bone marrow, colon, heart, liver, lymphoid, synovial, testicular, and thyroid tissues. In a sub-set of these cases, B19V capsid mRNA or proteins have been associated with increased inflammatory-related gene expression. The development of standard protocols to assay for B19V infection and expression in the context of non-erythroid, non-acute disease is warranted, and with further targeted studies, may begin to elucidate the impact of persistent B19V infection in vivo. These studies may determine the most conducive cellular environment for persistent gene expression and possible impact on disease pathogenesis.

Persistent Adeno-associated Virus 2 and Parvovirus B19 Sequences in Post-mortem Human Cerebellum

We previously reported in a large cohort (N = 104) of post-mortem tissues the detection of both the non-pathogenic adeno-associated virus (AAV2) in approximately 13% and the pathogenic human parvovirus B19 (B19) in approximately 42% of human brains, particularly the dorsolateral prefrontal cortex. Multiple animal parvoviruses target the developing cerebellum (CBLM) resulting in hypoplasia and ataxia, but very little is known about the human parvoviruses and their ability to infect or cause disease in the CBLM. We have now confirmed in the above cohort the presence of AAV2 and B19 sequences in the CBLM. Our results show that approximately 27% and approximately 70% of human CBLM are positive by nested polymerase chain reaction for AAV2 and B19 sequences, respectively. We also document in a second cohort (N = 10) the presence of AAV2 (50%) and B19 (100%) sequences in the CBLM and correlate our results for B19 with studies from matched sera. Eighty percent (80%) of this cohort was positive for anti-B19 IgG, while none were IgM+, suggesting that most individuals had been previously infected with B19 but none acutely. To our knowledge, this study is the first to demonstrate that both AAV2 and B19 sequences are present at relatively high frequencies in the CBLM and are likely due to persistent rather than acute infection. Further studies will lead to insights into AAV2- and/or B19-CBLM interactions including mechanisms of infection, persistence, and possibly neuropathology, including cerebellar hypoplasia and ataxia.

Detection of Parvovirus B19 Capsid Proteins in Testicular Tissues

OBJECTIVE To detect B19 capsid proteins, VP1 and VP2, in testicular tissues, both normal and tumor, using immunohistochemistry. METHODS Samples of normal, fetal, and tumor testicular tissue (n = 31) and normal testicular DNA (n = 1) were tested for the presence of B19. Immunohistochemistry staining was used for the detection of viral capsid proteins VP1 and VP2. Polymerase chain reaction with 4 primer sets was used to test for the presence of B19 DNA in a normal testicular sample. RESULTS B19 capsid protein VP1 and VP2 was detected by immunohistochemistry in 6 (85.7%) of 7 normal testicular samples and 17 (73.9%) of 23 tumor samples. The findings from a normal fetal testicular sample were equivocal. B19 DNA was detected in normal testicular DNA with 4 of the 4 primer sets used. CONCLUSION In contrast to previous reports, B19 capsid proteins VP1 and VP2 have now been detected in both normal and tumor testicular tissue. The persistence of B19 in a diverse range of tissues, including the testes, requires more research into the molecular mechanisms by which B19 can enter these cells, as well as the possible etiologic roles in chronic diseases, including cancer.

Increased IL-6 detection in adult and pediatric lymphoid tissue harboring Parvovirus B19

BACKGROUND Parvovirus B19 (B19V) is a common pathogenic virus infecting humans. Previous studies have shown evidence of B19V infection in patients with non-Hodgkins lymphoma (NHL) and Hodgkins lymphoma using ELISA and PCR on serum specimens. B19V nonstructural protein is known to alter the expression of cellular factors including interleukin-6 (IL-6), which can increase the risk for and worsen the prognosis of lymphomas. OBJECTIVE The objective of this study was to detect B19V capsid protein and IL-6 expression in normal and malignant lymphoid tissue, as well as any correlation between the two. STUDY DESIGN IHCs for B19V capsid protein, IL-6, and B19V co-receptors P-antigen and α5β1 integrin were performed on a tissue array containing 70 duplicated pediatric and adult lymphoma tissues and 5 duplicated benign lymph node sections. Cases were identified as normal, B-cell NHL, diffuse large B-cell NHL, Hodgkins lymphoma, extranodal NK/T cell lymphoma, anaplastic large cell lymphoma, or mantle cell lymphoma. IL-6 and B19V capsid staining were quantified using a positive pixel count algorithm, and P-antigen and α5β1 staining using a membrane quantification algorithm. RESULTS B19V capsid protein was detected in both benign and malignant lymphoid tissue. The Spearman rank correlation coefficient analysis was performed to determine the relationship between the level of positivity for B19V and IL-6 staining, yielding an overall correlation coefficient of 0.679 (p-value<0.0001). CONCLUSIONS Our results show a moderate correlation between the levels of positive B19V and IL-6 staining by IHC, indicating a possible role for B19V in the pathogenesis of lymphomas.

Human parvovirus B19 infection leads to downregulation of thyroid, estrogen, and retinoid hormone receptor expression.

Erythrovirus B19 (B19V) is a member of the family Parvoviridae. Infection with B19V has been linked to a variety of diseases including erythroid, thyroid, neurological and autoimmune diseases. Here we show that infection of primary CD36+ cells with B19V coincides with downregulation of thyroid, retinoid, and estrogen hormone receptors. In addition we show changes in expression of a variety of related downstream signaling genes participating in cancer and cardiac-related diseases in B19V-infected erythroid primary cells.

Trends in performance on the psychiatry resident-in-training examination (PRITE®): 10 years of data from a single institution.

ObjectiveThe purpose of this study was to examine trends in the Psychiatry Resident-In-Training Examination (PRITE®) scores at one institution from 2001 to 2010. The authors hypothesized that two factors, the 2003 implementation of the Accreditation Council for Graduate Medical Education (ACGME) duty-hour restrictions and the residency program’s 2008 restructuring of its curriculum to a half-day per week of didactics, would lead to improved scores.MethodResidents in the general psychiatry program at the University of Florida College of Medicine from 2001 to 2010 were included in this study. To examine the effect of the 2003 ACGME duty-hours change, the authors compared test results from 2001–2002 and 2003–2010. To examine the effect of the 2008 didactic restructuring, they compared test results from 2001–2007 and 2008–2010.ResultsThere were 288 PRITE test scores from 2001 to 2010 The authors did not find a statistical difference between test results before and after the 2003 implementation of ACGME duty-hour restrictions or between test results before and after the 2008 restructuring of residency didactics.ConclusionsThe hypothesis was rejected. The results of the literature review propose that examination scores are affected by other elements of residency training.

How Do Viruses Really Cause Neuropathology?: Is There More to the Story

Our work has focused over the years on Primate Erythroparvovirus 1 [more commonly known as parvovirus B19 (B19)]. B19 is a human pathogen that is the causative agent of erythema infectiosum, the common rash disease of childhood. In addition to several other human diseases, B19 has also been associated with multiple brain diseases. Those brain diseases include, but are not limited to, encephalitis, encephalopathy, meningitis, meningoencephalitis, ataxia, seizures, and stroke. To date, the association has grown, but causation has not been determined. Several possible mechanisms of pathogenesis including direct infection, indirect/epigenetic, reactivation, and autoimmune/ inflammatory cytotoxicity, as well as effects due to infection of other organs have been suggested. We will also offer other hypotheses.