Improved cell-specificity of adeno-associated viral vectors for medullary thyroid carcinoma using calcitonin gene regulatory elements.

Targeted gene therapy using recombinant adeno-associated virus (rAAV) vectors is a potential therapeutic strategy for treating cancer, and tissue-specific promoters may help with tissue targeting. Medullary thyroid carcinoma (MTC) is a disease of the calcitonin secreting thyroid C cells, and calcitonin is highly expressed in MTC tumors compared to other cells. To target MTC cells, we evaluated an rAAV serotype 2 vector (rAAV2-pM+104-GFP) containing a modified calcitonin/calcitonin gene related peptide promoter (pM+104) and a green fluorescent protein (GFP) reporter gene. In vitro transduction experiments comparing the MTC TT cell line with non-MTC cell lines demonstrated that rAAV2-pM+104-GFP infection yielded significantly (p < 0.05) higher GFP expression in TT cells than in non-MTC cell lines (HEK293 and HeLa), and significantly higher expression than in TT cells infected with the positive control rAAV2-pCBA-GFP vector. The rAAV2-pCBA-GFP control vector included a well-characterized, ubiquitously expresses control promoter, the chicken beta actin promoter with a cytomegalovirus enhancer (pCBA). In vivo experiments using a TT cell xenograft tumor mouse model showed that tumors directly injected with 2 x 1010 vg of rAAV2-pM+104-GFP vector resulted in GFP expression detected in 21.7% of cells, 48 hours after the injection. Furthermore, GFP expression was significantly higher for rAAV-pM+104-GFP treatments with a longer vector treatment duration and higher vector dose, with up to 52.6% (q < 0.05) GFP cells detected 72 hours after injecting 1x 1011 vg/tumor. These data show that we have developed an rAAV vector with improved selectivity for MTC.

Parvovirus and thyroid cancer.

Parvoviruses are some of the smallest DNA viruses known to infect a wide range of animal species and humans. Though not all parvoviruses are pathogenic, some can cause disease ranging from asymptomatic to benign to life-threatening. Recently, there has been an interest in the possible role of parvoviruses in thyroid disease in general. The objectives of this review are to cite and appraise the available evidence on the role of parvoviruses in thyroid cancer in particular. Little to no evidence is available directly linking animal parvoviruses and thyroid cancer, but there is a growing literature on the human erythrovirus B19 (EVB19) and its association with thyroid cancer. Of particular interest is the persistence and expression of EVB19 DNA, RNA, and protein in a wide variety of thyroid tissues. While a causative role of EVB19 in the pathogenesis of thyroid cancer cannot be supported at this time, an indirect role is hypothesized and discussed but with the recognition that the data are limited. Further studies are clearly warranted to determine the exact, if any, role of this human pathogen in thyroid cancer.

Parvovirus b19 persistence in abnormal thyroid tissue of a mature cystic ovarian teratoma: a case report.

Ovarian teratomas represent the most common neoplasm derived from germ cells and can contain mature ectodermal, mesodermal, and endodermal tissues. In rare cases, these teratomas can be composed predominantly or solely of thyroid tissue. These thyroid cells often function similarly to normal thyroid tissues. This laboratory and others have previously shown that parvovirus B19 (B19V) persists in primary and metastatic thyroid tissues. No reports exist on possible B19V persistence in thyroid tissues that may arise de novo outside the thyroid gland proper. In this case report, the detection of B19V (genotype 1) in the thyroid epithelial cells of a mature teratoma is reported. Nested PCR and immunohistochemistry were used to detect viral nucleic acids and proteins, respectively. Viral genomes were amplified in lesion DNA, confirming persistence of B19V. Positive immunohistochemical staining was seen for B19V capsid proteins in the thyroid epithelial cells within the mature teratoma, but not in surrounding ovarian tissue or in the non-thyroidal elements of the mature teratoma. These results demonstrate for the first time that thyroid epithelial cells, derived from non-thyroid tissue, are capable of supporting B19V infection and persistence.

Infection and persistence of erythrovirus B19 in benign and cancerous thyroid tissues.

Human erythrovirus B19 (EVB19) is a small, pathogenic DNA virus that has been associated with a wide range of illnesses. The primary site of replication is in bone marrow-derived erythroid progenitor cells, but EVB19 DNA has been detected in a wide range of organs. Recently, studies have linked EVB19 to thyroid cancers and other thyroid diseases. Previous studies from multiple laboratories have detected EVB19 capsid proteins in Graves' disease, Hashimoto's thyroiditis, and thyroid cancer tissues. Data on viral gene expression and mechanism of infection in the thyroid are lacking. To investigate EVB19 infection and persistence in the thyroid, previously archived adult and pediatric tissue sections were examined for EVB19 DNA, RNA, and capsid proteins, as well as EVB19 receptor P-antigen and co-receptor α5ß1 integrin. EVB19 DNA and protein were detected in a majority of tissues examined (87% and 68%, respectively). Detection was similar in adult and pediatric samples. Quantification of viral genomes revealed no significant difference in the amount of viral DNA in benign, cancerous, or metastatic thyroid tissues. EVB19 capsid RNA was detected in 67% of the tissues examined, confirming at least low-level viral gene expression. Immunohistochemical staining for P-antigen and α5ß1 detected the receptor and co-receptor most frequently on normal thyroid epithelial cells. EVB19 capsid staining could be detected in tumors lacking viral receptors. These results suggest that normal thyroid epithelial cells are the initial target for EVB19 infection in the thyroid and allow for continued persistence in both normal and cancerous thyroid tissues.

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.

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-Hodgkin's lymphoma (NHL) and Hodgkin's 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 α5ß1 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, Hodgkin's 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 α5ß1 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.

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.

Parvovirus B19 infection in Hashimoto's thyroiditis, papillary thyroid carcinoma, and anaplastic thyroid carcinoma.

BACKGROUND: The human pathogenic parvovirus B19 (B19) has recently been detected in papillary thyroid carcinoma (PTC) and Hashimoto's thyroiditis (HT) tissues at a high frequency in two studies of a Chinese cohort. We wanted to extend these data to include another cohort and expand the thyroid tumor tissue types assessed. In particular, we were interested to find whether B19 also infects anaplastic thyroid carcinoma (ATC), one of the most aggressive human cancers. METHODS: Commercially available thyroid tumor tissue arrays were used to detect B19 capsid protein by immunohistochemistry in various types of thyroid tumors and disorders. The arrays were representative of the four main types of thyroid tumors, as well as other thyroid autoimmune disorders such as HT and Graves' disease, and adenomas, goiters, lymphomas, and normal thyroid tissue. In total, at least 12 different types of thyroid conditions as well as normal tissue were represented, many with multiple subjects. RESULTS: Twenty-one of the 24 (88%) PTC tumors, 3 of the 3 ATC/undifferentiated tumors, and 3 of the 3 HT tissue samples were positive for B19 capsid protein by immunohistochemistry. The localization of the protein differed based on pathological disease type, with a nuclear to cytoplasmic shift seen from unaffected to tumor tissue. CONCLUSIONS: We extend the data available on B19 detection in the thyroid to show a high correlation of virus in another cohort of PTC and HT at the protein level. We also show, for the first time, B19 infection of much more highly aggressive ATC/undifferentiated tumors. Nuclear to cytoplasmic shift in B19 protein in cancer tissue suggests a possible link between B19 and thyroid cancer pathogenesis/progression.

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.

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.

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 approximately 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 approximately 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.

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.

Requirement of caspase-3 for efficient apoptosis induction and caspase-7 activation but not viral replication or cell rounding in cells infected with vesicular stomatitis virus.

Infection with vesicular stomatitis virus (VSV), a rhabdovirus and economically significant animal pathogen, was previously demonstrated to induce apoptosis. The mechanism of induction and the role of apoptosis in the VSV-host response have not been completely elucidated. Previous data from our laboratory have suggested that caspase-3 is required for the induction of apoptosis but not viral replication in VSV-infected cells. However, these studies used inhibitors that are selective but not specific for caspase-3. To circumvent this difficulty, we infected both MCF-7 cells which do not express caspases-3 (null), and stable transfectants which express caspase-3 (C3+). When caspase-3 null cells were infected, significant PARP cleavage did not occur, but when C3+ cells were infected, PARP cleavage did occur efficiently. Studies in null and C3+ also suggest that: (1) caspases-3 and -7 are activated sequentially after VSV infection; (2) cell shrinkage and detachment are caspase-3 dependent, but cell rounding is not; and (3) the viral titers were similar between caspase-3 null and C3+ cells suggesting that activation of caspases-3 and -7 are not required for viral replication. Taken together, these results strongly support that the activation of caspase-3 by VSV infection is required for efficient apoptosis induction but not viral replication in vitro. Apoptosis mediated by caspase-3, then, is likely either a host cell response to viral replication or perhaps may be required for in vivo viral replication and spread.