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author:("Bryan, canine")
1.  Protection Provided by a Herpes Simplex Virus 2 (HSV-2) Glycoprotein C and D Subunit Antigen Vaccine against Genital HSV-2 Infection in HSV-1-Seropositive Guinea Pigs 
Journal of Virology  2014;88(4):2000-2010.
A prophylactic vaccine for genital herpes disease remains an elusive goal. We report the results of two studies performed collaboratively in different laboratories that assessed immunogenicity and vaccine efficacy in herpes simplex virus 1 (HSV-1)-seropositive guinea pigs immunized and subsequently challenged intravaginally with HSV-2. In study 1, HSV-2 glycoproteins C (gC2) and D (gD2) were produced in baculovirus and administered intramuscularly as monovalent or bivalent vaccines with CpG and alum. In study 2, gD2 was produced in CHO cells and given intramuscularly with monophosphoryl lipid A (MPL) and alum, or gC2 and gD2 were produced in glycoengineered Pichia pastoris and administered intramuscularly as a bivalent vaccine with Iscomatrix and alum to HSV-1-naive or -seropositive guinea pigs. In both studies, immunization boosted neutralizing antibody responses to HSV-1 and HSV-2. In study 1, immunization with gC2, gD2, or both immunogens significantly reduced the frequency of genital lesions, with the bivalent vaccine showing the greatest protection. In study 2, both vaccines were highly protective against genital disease in naive and HSV-1-seropositive animals. Comparisons between gD2 and gC2/gD2 in study 2 must be interpreted cautiously, because different adjuvants, gD2 doses, and antigen production methods were used; however, significant differences invariably favored the bivalent vaccine. Immunization of naive animals with gC2/gD2 significantly reduced the number of days of vaginal shedding of HSV-2 DNA compared with that for mock-immunized animals. Surprisingly, in both studies, immunization of HSV-1-seropositive animals had little effect on recurrent vaginal shedding of HSV-2 DNA, despite significantly reducing genital disease.
PMCID: PMC3911559  PMID: 24284325
2.  Molecular Signatures of Recurrent Hepatocellular Carcinoma Secondary to Hepatitis C Virus following Liver Transplantation 
Journal of Transplantation  2013;2013:878297.
Chronic hepatitis C virus (HCV) induced hepatocellular carcinoma (HCC) is a primary indication for liver transplantation (LT). In western countries, the estimated rate of HCC recurrence following LT is between 15% and 20% and is a major cause of mortality. Currently, there is no standard method to treat patients who are at high risk for HCC recurrence. The aim of this study was to investigate the molecular signatures underlying HCC recurrence that may lead to future studies on gene regulation contributing to new therapeutic options. Two groups of patients were selected, one including patients with HCV who developed HCC recurrence (HCC-R) ≤3 years from LT and the second group including patients with HCV who did not have recurrent HCC (HCC-NR). Microarray analysis containing more than 29,000 known genes was performed on formalin-fixed-paraffin-embedded (FFPE) liver tissue from explanted livers. Gene expression profiling revealed 194 differentially regulated genes between the two groups. These genes belonged to cellular networks including cell cycle G1/S checkpoint regulators, RAN signaling, chronic myeloid leukemia signaling, molecular mechanisms of cancer, FXR/RXR activation and hepatic cholestasis. A subset of molecular signatures associated with HCC recurrence was found. The expression levels of these genes were validated by quantitative PCR analysis.
PMCID: PMC3860124  PMID: 24377043
3.  Humoral, Mucosal, and Cell-Mediated Immunity Against Vaccine and Nonvaccine Genotypes After Administration of Quadrivalent Human Papillomavirus Vaccine to HIV-Infected Children 
The Journal of Infectious Diseases  2012;206(8):1309-1318.
Objectives. To characterize the immunogenicity of a quadrivalent human papillomavirus vaccine (QHPV) in human immunodeficiency virus (HIV)–infected children, we studied their immune responses to 3 or 4 doses.
Methods. HIV-infected children aged 7–12 years with a CD4 cell percentage of ≥15% of lymphocytes, received 3 doses of QHPV with or without a fourth dose after 72 weeks. Type-specific and cross-reactive antibodies and cell-mediated immunity were measured.
Results. Type-specific antibodies to HPV6, 11, and 16 were detected in 100% and ≥94% of children at 4 and 72 weeks, respectively, after the third QHPV dose. Corresponding numbers for HPV18 were 97% and 76%, respectively. A fourth QHPV dose increased seropositivity to ≥96% for all vaccine genotypes. Four weeks after the third QHPV dose, 67% of vaccinees seroconverted to HPV31, an HPV16-related genotype not in the vaccine; 69% and 39% of vaccinees developed mucosal HPV16 and 18 immunoglobulin G antibodies, respectively; and 60% and 52% of vaccinees developed cytotoxic T lymphocytes (CTLs) for HPV16 and 31, respectively.
Conclusions. Three QHPV doses generated robust and persistent antibodies to HPV6, 11, and 16 but comparatively weaker responses to HPV18. A fourth dose increased antibodies against all vaccine genotypes in an anamnestic fashion. CTLs and mucosal antibodies against vaccine genotypes, as well as cross-reactive antibodies and CTL against nonvaccine genotypes, were detected.
PMCID: PMC3529604  PMID: 22859825
4.  H5N1 Virus Causes Significant Perturbations in Host Proteome Very Early in Influenza Virus-Infected Primary Human Monocyte-Derived Macrophages 
The Journal of Infectious Diseases  2012;206(5):640-645.
H5N1 influenza viruses, which cause disease in humans, have unusually high pathogenicity. The temporal response of primary human monocyte-derived macrophages infected with highly pathogenic H5N1 and seasonal H1N1 influenza viruses was evaluated using mass spectrometry–based quantitative proteomic profiling. This was done in order to demonstrate significant perturbation of the host proteome upon viral infection, as early as 1 hour after infection. This early host response distinguished H5N1 infection from H1N1 infection, the latter inducing less of a response. The most pronounced effect was observed on the translational machinery, suggesting that H5N1 might gain advantage in replication by using the cell protein synthesis machinery early in the infection.
PMCID: PMC3491741  PMID: 22822004
5.  Chronic immune activation is a distinguishing feature of liver and PBMC gene signatures from HCV/HIV coinfected patients and may contribute to hepatic fibrogenesis 
Virology  2012;430(1):43-52.
Hepatitis C virus/human immunodeficiency virus (HCV/HIV) coinfected patients demonstrate accelerated progression to severe liver injury in comparison to HCV monoinfected patients, although the underlying mechanisms are unclear owing to infection of separate tissue compartments with two distinct viral pathogens. Microarray analysis of paired liver biopsy and peripheral blood mononuclear cell (PBMC) specimens from HCV/HIV coinfected and HCV monoinfected patients identified a gene expression signature associated with increased inflammation and immune activation that was present only in liver and PBMC samples from coinfected patients. We also identified in these samples liver- and PBMC-specific signatures enriched with fibrogenic/hepatic stellate activation and proinflammatory genes, respectively. Finally, Bayesian networks were constructed by assimilating these data with existing data from liver and PBMC samples from other cohorts, augmenting enrichment of biologically important pathways and further indicating that chronic immune activation in HCV/HIV coinfection may exacerbate liver disease progression in coinfected patients.
PMCID: PMC3371131  PMID: 22608059
hepatitis C virus; human immunodeficiency virus; HCV/HIV coinfection; liver; peripheral blood; hepatic fibrosis; systems biology; gene expression; pathogenesis
7.  Evidence of immune memory 8.5 years following administration of a prophylactic human papillomavirus type 16 vaccine 
Journal of Clinical Virology  2011;53(3):239-243.
The duration of protection conferred by prophylactic human papillomavirus (HPV) L1 virus-like particle vaccines is a critical determinant of their public health impact. A feature of vaccines that confer long-term immunity is their ability to induce immune memory.
We evaluated antibody responses against HPV types 6, 11, 16 and 18 following administration of the quadrivalent HPV-6/11/16/18 vaccine to women who had previously received a monovalent HPV-16 vaccine.
Study design
As part of an extended follow-up study conducted between 2006 and 2009 in Seattle, Washington, we administered the quadrivalent HPV-6/11/16/18 vaccine to 52 women (19 vaccine and 33 placebo recipients) who had participated in a monovalent HPV-16 vaccine trial 8.5 years earlier. Serum samples were tested for anti-HPV antibodies using competitive Luminex immunoassay.
Following administration of the first dose of the quadrivalent HPV-6/11/16/18 vaccine, the anti-HPV-16 geometric mean titer among monovalent HPV-16 vaccine recipients (GMT = 5024.0 milli-Merck units per milliliter [mMU/mL]; 95% confidence interval [CI]: 2710.1, 9313.6 mMU/mL) substantially exceeded that among the placebo recipients (GMT = 136.1; 95% CI: 78.5, 235.8 mMU/mL; p < 0.01) and their own highest anti-HPV-16 response observed during the original trial (GMT at month 7 of the original trial = 1552.7 mMU/mL; 95% CI: 1072.6, 2247.7 mMU/mL; p < 0.01).
The findings suggest that the administration of the three-dose regimen of the monovalent HPV-16 vaccine had produced memory lymphocytes, characterized by a heightened immune response following administration of the quadrivalent HPV-6/11/16/18 vaccine that effectively served as an antigen challenge.
PMCID: PMC3279625  PMID: 22209292
Human papillomavirus type 16; Vaccines; Immune memory
8.  Modeling Host Genetic Regulation of Influenza Pathogenesis in the Collaborative Cross 
PLoS Pathogens  2013;9(2):e1003196.
Genetic variation contributes to host responses and outcomes following infection by influenza A virus or other viral infections. Yet narrow windows of disease symptoms and confounding environmental factors have made it difficult to identify polymorphic genes that contribute to differential disease outcomes in human populations. Therefore, to control for these confounding environmental variables in a system that models the levels of genetic diversity found in outbred populations such as humans, we used incipient lines of the highly genetically diverse Collaborative Cross (CC) recombinant inbred (RI) panel (the pre-CC population) to study how genetic variation impacts influenza associated disease across a genetically diverse population. A wide range of variation in influenza disease related phenotypes including virus replication, virus-induced inflammation, and weight loss was observed. Many of the disease associated phenotypes were correlated, with viral replication and virus-induced inflammation being predictors of virus-induced weight loss. Despite these correlations, pre-CC mice with unique and novel disease phenotype combinations were observed. We also identified sets of transcripts (modules) that were correlated with aspects of disease. In order to identify how host genetic polymorphisms contribute to the observed variation in disease, we conducted quantitative trait loci (QTL) mapping. We identified several QTL contributing to specific aspects of the host response including virus-induced weight loss, titer, pulmonary edema, neutrophil recruitment to the airways, and transcriptional expression. Existing whole-genome sequence data was applied to identify high priority candidate genes within QTL regions. A key host response QTL was located at the site of the known anti-influenza Mx1 gene. We sequenced the coding regions of Mx1 in the eight CC founder strains, and identified a novel Mx1 allele that showed reduced ability to inhibit viral replication, while maintaining protection from weight loss.
Author Summary
Host responses to an infectious agent are highly variable across the human population, however, it is not entirely clear how various factors such as pathogen dose, demography, environment and host genetic polymorphisms contribute to variable host responses and infectious outcomes. In this study, a new in vivo experimental model was used that recapitulates many of the genetic characteristics of an outbred population, such as humans. By controlling viral dose, environment and demographic variables, we were able to focus on the role that host genetic variation plays in influenza virus infection. Both the range of disease phenotypes and the combinations of sets of disease phenotypes at 4 days post infection across this population exhibited a large amount of diversity, reminiscent of the variation seen across the human population. Multiple host genome regions were identified that contributed to different aspects of the host response to influenza infection. Taken together, these results emphasize the critical role of host genetics in the response to infectious diseases. Given the breadth of host responses seen within this population, several new models for unique host responses to infection were identified.
PMCID: PMC3585141  PMID: 23468633
9.  Efficacy of Quadrivalent HPV Vaccine against HPV Infection and Disease in Males 
The New England journal of medicine  2011;364(5):401-411.
Infection with human papillomavirus (HPV) and diseases caused by HPV are common in boys and men. We report on the safety of a quadrivalent vaccine (active against HPV types 6, 11, 16, and 18) and on its efficacy in preventing the development of external genital lesions and anogenital HPV infection in boys and men.
We enrolled 4065 healthy boys and men 16 to 26 years of age, from 18 countries in a randomized, placebo-controlled, double-blind trial. The primary efficacy objective was to show that the quadrivalent HPV vaccine reduced the incidence of external genital lesions related to HPV-6, 11, 16, or 18. Efficacy analyses were conducted in a per-protocol population, in which subjects received all three vaccinations and were negative for relevant HPV types at enrollment, and in an intention-to-treat population, in which subjects received vaccine or placebo, regardless of baseline HPV status.
In the intention-to-treat population, 36 external genital lesions were seen in the vaccine group as compared with 89 in the placebo group, for an observed efficacy of 60.2% (95% confidence interval [CI], 40.8 to 73.8); the efficacy was 65.5% (95% CI, 45.8 to 78.6) for lesions related to HPV-6, 11, 16, or 18. In the per-protocol population, efficacy against lesions related to HPV-6, 11, 16, or 18 was 90.4% (95% CI, 69.2 to 98.1). Efficacy with respect to persistent infection with HPV-6, 11, 16, or 18 and detection of related DNA at any time was 47.8% (95% CI, 36.0 to 57.6) and 27.1% (95% CI, 16.6 to 36.3), respectively, in the intention-to-treat population and 85.6% (97.5% CI, 73.4 to 92.9) and 44.7% (95% CI, 31.5 to 55.6) in the per-protocol population. Injection-site pain was significantly more frequent among subjects receiving quadrivalent HPV vaccine than among those receiving placebo (57% vs. 51%, P<0.001).
Quadrivalent HPV vaccine prevents infection with HPV-6, 11, 16, and 18 and the development of related external genital lesions in males 16 to 26 years of age. (Funded by Merck and others; number, NCT00090285.)
PMCID: PMC3495065  PMID: 21288094
10.  Live Attenuated Herpes Simplex Virus 2 Glycoprotein E Deletion Mutant as a Vaccine Candidate Defective in Neuronal Spread 
Journal of Virology  2012;86(8):4586-4598.
A herpes simplex virus 2 (HSV-2) glycoprotein E deletion mutant (gE2-del virus) was evaluated as a replication-competent, attenuated live virus vaccine candidate. The gE2-del virus is defective in epithelial cell-to-axon spread and in anterograde transport from the neuron cell body to the axon terminus. In BALB/c and SCID mice, the gE2-del virus caused no death or disease after vaginal, intravascular, or intramuscular inoculation and was 5 orders of magnitude less virulent than wild-type virus when inoculated directly into the brain. No infectious gE2-del virus was recovered from dorsal root ganglia (DRG) after multiple routes of inoculation; however, gE2-del DNA was detected by PCR in lumbosacral DRG at a low copy number in some mice. Importantly, no recurrent vaginal shedding of gE2-del DNA was detected in immunized guinea pigs. Intramuscular immunization outperformed subcutaneous immunization in all parameters evaluated, although individual differences were not significant, and two intramuscular immunizations were more protective than one. Immunized animals had reduced vaginal disease, vaginal titers, DRG infection, recurrent genital lesions, and recurrent vaginal shedding of HSV-2 DNA; however, protection was incomplete. A combined modality immunization using live virus and HSV-2 glycoprotein C and D subunit antigens in guinea pigs did not totally eliminate recurrent lesions or recurrent vaginal shedding of HSV-2 DNA. The gE2-del virus used as an immunotherapeutic vaccine in previously HSV-2-infected guinea pigs greatly reduced the frequency of recurrent genital lesions. Therefore, the gE2-del virus is safe, other than when injected at high titer into the brain, and is efficacious as a prophylactic and immunotherapeutic vaccine.
PMCID: PMC3318599  PMID: 22318147
11.  Immunization with a Vaccine Combining Herpes Simplex Virus 2 (HSV-2) Glycoprotein C (gC) and gD Subunits Improves the Protection of Dorsal Root Ganglia in Mice and Reduces the Frequency of Recurrent Vaginal Shedding of HSV-2 DNA in Guinea Pigs Compared to Immunization with gD Alone ▿  
Journal of Virology  2011;85(20):10472-10486.
Attempts to develop a vaccine to prevent genital herpes simplex virus 2 (HSV-2) disease have been only marginally successful, suggesting that novel strategies are needed. Immunization with HSV-2 glycoprotein C (gC-2) and gD-2 was evaluated in mice and guinea pigs to determine whether adding gC-2 to a gD-2 subunit vaccine would improve protection by producing antibodies that block gC-2 immune evasion from complement. Antibodies produced by gC-2 immunization blocked the interaction between gC-2 and complement C3b, and passive transfer of gC-2 antibody protected complement-intact mice but not C3 knockout mice against HSV-2 challenge, indicating that gC-2 antibody is effective, at least in part, because it prevents HSV-2 evasion from complement. Immunization with gC-2 also produced neutralizing antibodies that were active in the absence of complement; however, the neutralizing titers were higher when complement was present, with the highest titers in animals immunized with both antigens. Animals immunized with the gC-2-plus-gD-2 combination had robust CD4+ T-cell responses to each immunogen. Multiple disease parameters were evaluated in mice and guinea pigs immunized with gC-2 alone, gD-2 alone, or both antigens. In general, gD-2 outperformed gC-2; however, the gC-2-plus-gD-2 combination outperformed gD-2 alone, particularly in protecting dorsal root ganglia in mice and reducing recurrent vaginal shedding of HSV-2 DNA in guinea pigs. Therefore, the gC-2 subunit antigen enhances a gD-2 subunit vaccine by stimulating a CD4+ T-cell response, by producing neutralizing antibodies that are effective in the absence and presence of complement, and by blocking immune evasion domains that inhibit complement activation.
PMCID: PMC3187515  PMID: 21813597
12.  Expression Quantitative Trait Loci for Extreme Host Response to Influenza A in Pre-Collaborative Cross Mice 
G3: Genes|Genomes|Genetics  2012;2(2):213-221.
Outbreaks of influenza occur on a yearly basis, causing a wide range of symptoms across the human population. Although evidence exists that the host response to influenza infection is influenced by genetic differences in the host, this has not been studied in a system with genetic diversity mirroring that of the human population. Here we used mice from 44 influenza-infected pre-Collaborative Cross lines determined to have extreme phenotypes with regard to the host response to influenza A virus infection. Global transcriptome profiling identified 2671 transcripts that were significantly differentially expressed between mice that showed a severe (“high”) and mild (“low”) response to infection. Expression quantitative trait loci mapping was performed on those transcripts that were differentially expressed because of differences in host response phenotype to identify putative regulatory regions potentially controlling their expression. Twenty-one significant expression quantitative trait loci were identified, which allowed direct examination of genes associated with regulation of host response to infection. To perform initial validation of our findings, quantitative polymerase chain reaction was performed in the infected founder strains, and we were able to confirm or partially confirm more than 70% of those tested. In addition, we explored putative causal and reactive (downstream) relationships between the significantly regulated genes and others in the high or low response groups using structural equation modeling. By using systems approaches and a genetically diverse population, we were able to develop a novel framework for identifying the underlying biological subnetworks under host genetic control during influenza virus infection.
PMCID: PMC3284329  PMID: 22384400
eQTL; influenza; collaborative cross; host response; SEM; Mouse Collaborative Cross; Mouse Genetic Resource
13.  Comparison of Real-Time Multiplex Human Papillomavirus (HPV) PCR Assays with the Linear Array HPV Genotyping PCR Assay and Influence of DNA Extraction Method on HPV Detection▿‡ 
Journal of Clinical Microbiology  2011;49(5):1899-1906.
Real-time human papillomavirus (HPV) type-specific multiplex PCR assays were developed to detect HPV DNA in specimens collected for the efficacy determination of the quadrivalent HPV (type 6, 11, 16, and 18) L1 virus-like particle (VLP) vaccine (Gardasil). We evaluated the concordance between type-specific multiplex HPV PCR and the widely used, commercially available Roche Linear Array genotyping PCR assay. Female genital swab specimens were tested for the presence of L1, E6, and E7 sequences of HPV type 6 (HPV6), HPV11, HPV16, HPV18, HPV31, HPV45, HPV52, and HPV58 and E6 and E7 sequences of HPV33, HPV35, HPV39, HPV51, HPV56, and HPV59 in type- and gene-specific real-time multiplex PCR assays. Specimens were also tested for the presence of L1 sequences using two versions of the Roche Linear Array genotyping assay. Measures of concordance of a modified version of the Linear Array and the standard Linear Array PCR assay were evaluated. With specimen DNA extraction using the Qiagen Spin blood kit held as the constant, multiplex PCR assays detect more HPV-positive specimens for the 14 HPV types common to both than either version of the Linear Array HPV genotyping assay. Type-specific agreements between the assays were good, at least 0.838, but were often driven by negative agreement in HPV types with low prevalence, as evidenced by reduced proportions of positive agreement. Overall HPV status agreements ranged from 0.615 for multiplex PCR and standard Linear Array to 0.881 for multiplex PCR and modified Linear Array. An alternate DNA extraction technique, that used by the Qiagen MinElute kit, impacted subsequent HPV detection in both the multiplex PCR and Linear Array assays.
PMCID: PMC3122643  PMID: 21346041
14.  Comparison of Real-Time Multiplex Human Papillomavirus (HPV) PCR Assays with INNO-LiPA HPV Genotyping Extra Assay▿ 
Journal of Clinical Microbiology  2011;49(5):1907-1912.
Real-time type-specific multiplex human papillomavirus (HPV) PCR assays were developed to detect HPV DNA in samples collected for the efficacy determination of the quadrivalent HPV (type 6, 11, 16, and 18) L1 virus-like particle (VLP) vaccine (Gardasil). Additional multiplex (L1, E6, and E7 open reading frame [ORF]) or duplex (E6 and E7 ORF) HPV PCR assays were developed to detect high-risk HPV types, including HPV type 31 (HPV31), HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV56, HPV58, and HPV59. Here, we evaluated clinical specimen concordance and compared the limits of detection (LODs) between multiplex HPV PCR assays and the INNO-LiPA HPV Genotyping Extra assay, which detects 28 types, for the 14 HPV types common to both of these methods. Overall HPV detection agreement rates were >90% for swabs and >95% for thin sections. Statistically significant differences in detection were observed for HPV6, HPV16, HPV18, HPV35, HPV39, HPV45, HPV56, HPV58, and HPV59 in swabs and for HPV45, HPV58, and HPV59 in thin sections. Where P was <0.05, discordance was due to detection of more HPV-positive samples by the multiplex HPV PCR assays. LODs were similar for eight HPV types, significantly lower in multiplex assays for five HPV types, and lower in INNO-LiPA for HPV6 only. LODs were under 50 copies for all HPV types, with the exception of HPV39, HPV58, and HPV59 in the INNO-LiPA assay. The overall percent agreement for detection of 14 HPV types between the type-specific multiplex HPV PCR and INNO-LiPA genotyping assays was good. The differences in positive sample detection favored multiplex HPV PCR, suggesting increased sensitivity of HPV DNA detection by type-specific multiplex HPV PCR assays.
PMCID: PMC3122697  PMID: 21068278
15.  Human papillomavirus type 16 modifies the association between fruit consumption and head and neck squamous cell carcinoma 
Human papillomavirus type-16 (HPV16) is a risk factor for head and neck squamous cell carcinoma (HNSCC). HPV-positive cancers have distinct disease cofactors and improved survival following treatment. There is conflicting evidence of a protective association of fruit consumption with HNSCC. As HPV-related disease is clinically distinct, we investigated whether the association between fruit consumption and HNSCC risk was modified by exposure to HPV16. We studied 270 cases and 493 controls with fruit intake information and known HPV16 antibody status. Cases were identified at nine Boston-area medical facilities between 1999 and 2003. Controls were randomly selected from the greater population and frequency-matched to cases by age, gender, and town of residence. Controlling for age, gender, race, smoking, alcohol, total energy intake, body mass index and education, the seronegative individuals had a significantly lower risk of HNSCC with increasing total fruit consumption (ORtertile 2: 0.60, (95% CI 0.38, 0.95); ORtertile 3: 0.57, (0.35, 0.95)) and, specifically, increasing citrus fruit consumption (ORtertile 2: 0.61, (0.39, 0.97); ORtertile 3: 0.59, (0.37, 0.96)). However, among the seropositive, risk increased with greater fruit consumption (ORtertile 2: 2.27, (0.92, 5.58); ORtertile 3: 1.40, (0.55, 3.59) and citrus fruit consumption (ORtertile 2: 3.35, (1.36, 8.24); ORtertile 3: 3.15, (1.23, 8.08)). This interaction was statistically significant (p-value <0.05), showing that fruit consumption was associated with a reduced HNSCC risk among HPV16-seronegative individuals but an increased HNSCC risk among the HPV16-seropositive. These findings suggest that dietary factors dramatically alter the pattern of occurrence of HPV-associated HNSCC and show that viral-related disease is clinically and etiologically distinct.
PMCID: PMC3122126  PMID: 19064557
Human papillomavirus (HPV); head and neck squamous cell carcinoma (HNSCC); citrus fruit; vitamin C; effect modification
16.  Longer-term efficacy of a prophylactic monovalent human papillomavirus type 16 vaccine 
Vaccine  2009;27(41):5612-5619.
We conducted an extended follow-up study (March 2006 – May 2008) to assess the longer-term efficacy of a prophylactic monovalent human papillomavirus (HPV) type 16 L1 virus-like particle vaccine in women (n = 290) who had enrolled in a randomized controlled trial of this vaccine during October 1998 – November 1999 in Seattle and remained HPV-16 DNA negative during the course of that trial. During the extended follow-up period, none of the vaccine recipients was found to be infected with HPV-16 or developed HPV-16-related cervical lesions; among placebo recipients, 6 women were found to be infected with HPV-16 (vaccine efficacy [VE] = 100%; 95% confidence interval [CI]: 29% – 100%) and 3 women developed HPV-16-related cervical lesions (VE = 100%; 95% CI: <0% – 100%). Approximately 86% of vaccine recipients remained HPV-16 competitive Luminex immunoassay seropositive at an average of 8.5 years of follow-up. During the combined original trial and extended follow-up period, 20 and 22 women developed any cervical lesion regardless of HPV type among the vaccine and placebo recipients, respectively (VE = 15%; 95% CI: <0% – 56%). The results suggest that this monovalent HPV-16 vaccine remains efficacious through 8.5 years after its administration.
PMCID: PMC2749988  PMID: 19647066
Human papillomavirus type 16; Prophylactic vaccines; Efficacy
17.  Genetic variation in the vitamin C transporter, SLC23A2, modifies the risk of HPV16-associated head and neck cancer 
Carcinogenesis  2009;30(6):977-981.
Human papillomavirus (HPV) type 16 infection is an etiologic factor in a subset of head and neck squamous cell carcinomas (HNSCC). It is unknown if host genetic susceptibility modifies the HPV16–HNSCC association. DNA samples collected as part of a Boston area case–control study of HNSCC were genotyped for single-nucleotide polymorphisms (SNPs) from the National Cancer Institute's SNP500Cancer database. Analysis of demographic, phenotypic and genotypic data for 319 HNSCC cases and 495 frequency-matched controls was performed using unconditional logistic regression. All reported P-values are two sided. We identified a polymorphism in the sodium-dependent vitamin C transporter SLC23A2 that modifies the risk of HNSCC associated with HPV16 infection. Among those with a wild-type allele at SLC23A2, the risk of HNSCC associated with HPV16-positive serology was 5.0 (95% confidence interval (CI) = 3.2–7.8). However, among those with a homozygous variant genotype, the risk of HNSCC associated with HPV16 was attenuated [odds ratio (OR) = 2.8; 95% CI = 1.2–6.2]. Further, when we tested whether genotype modified the interaction between citrus exposure, HPV16, and HNSCC, we found a dramatically increased risk of HNSCC for those with a wild-type SLC23A2 allele, HPV16-positive serology and high citrus intake (OR = 7.4; 95% CI = 3.6–15.1). These results suggest that SLC23A2 genetic variation alters HPV16-associated HNSCC while also highlighting the important role of citrus exposure in this disease.
PMCID: PMC2691140  PMID: 19346260
18.  Study Comparing Human Papillomavirus (HPV) Real-Time Multiplex PCR and Hybrid Capture II INNO-LiPA v2 HPV Genotyping PCR Assays ▿  
Journal of Clinical Microbiology  2009;47(7):2106-2113.
Human papillomavirus (HPV) DNA genotyping is an essential test to establish efficacy in HPV vaccine clinical trials and HPV prevalence in natural history studies. A number of HPV DNA genotyping methods have been cited in the literature, but the comparability of the outcomes from the different methods has not been well characterized. Clinically, cytology is used to establish possible HPV infection. We evaluated the sensitivity and specificity of HPV multiplex PCR assays compared to those of the testing scheme of the Hybrid Capture II (HCII) assay followed by an HPV PCR/line hybridization assay (HCII-LiPA v2). SurePath residual samples were split into two aliquots. One aliquot was subjected to HCII testing followed by DNA extraction and LiPA v2 genotyping. The second aliquot was shipped to a second laboratory, where DNA was extracted and HPV multiplex PCR testing was performed. Comparisons were evaluated for 15 HPV types common in both assays. A slightly higher proportion of samples tested positive by the HPV multiplex PCR than by the HCII-LiPA v2 assay. The sensitivities of the multiplex PCR assay relative to those of the HCII-LiPA v2 assay for HPV types 6, 11, 16, and 18, for example, were 0.806, 0.646, 0.920, and 0.860, respectively; the specificities were 0.986, 0.998, 0.960, and 0.986, respectively. The overall comparability of detection of the 15 HPV types was quite high. Analyses of DNA genotype testing compared to cytology results demonstrated a significant discordance between cytology-negative (normal) and HPV DNA-positive results. This demonstrates the challenges of cytological diagnosis and the possibility that a significant number of HPV-infected cells may appear cytologically normal.
PMCID: PMC2708522  PMID: 19420164
19.  The Dlx3 protein harbors basic residues required for nuclear localization, transcriptional activity and binding to Msx1 
Journal of cell science  2000;113(Pt 22):4013-4023.
The murine Dlx3 protein is a putative transcriptional activator that has been implicated during development and differentiation of epithelial tissue. Dlx3 contains a homeodomain and mutational analysis has revealed two regions, one N-terminal and one C-terminal to the homeodomain, that act as transcriptional activators in a yeast one-hybrid assay. In addition to transactivation, data are presented to demonstrate specific DNA binding and an association between Dlx3 and the Msx1 protein in vitro. Immunohistochemical analysis confirmed coexpression of Dlx3 and Msx1 proteins in the differentiated layers of murine epidermal tissues.
Transcription factor function requires nuclear localization. In this study, the intracellular localization of the green fluorescent protein fused to Dlx3 was examined in keratinocytes induced to differentiate by calcium and is shown to localize to the nucleus. A bipartite nuclear localization signal (NLS) was identified by mutational analysis and shown to be sufficient for nuclear localization. This was demonstrated by insertion of the Dlx3 bipartite NLS sequence into a cytoplasmic fusion protein, GFP-keratin 14, which functionally redirected GFP-keratin 14 expression to the nucleus. Further analysis of Dlx3 NLS mutants revealed that the Dlx3 NLS sequences are required for specific DNA binding, transactivation potential and interactions with the Msx1 protein.
PMCID: PMC1317294  PMID: 11058088
Dlx3; Homeodomain; Transactivation; DNA binding; Bipartite nuclear localization signal
20.  A Novel Staphylococcus aureus Vaccine: Iron Surface Determinant B Induces Rapid Antibody Responses in Rhesus Macaques and Specific Increased Survival in a Murine S. aureus Sepsis Model  
Infection and Immunity  2006;74(4):2215-2223.
Staphylococcus aureus is a major cause of nosocomial infections worldwide, and the rate of resistance to clinically relevant antibiotics, such as methicillin, is increasing; furthermore, there has been an increase in the number of methicillin-resistant S. aureus community-acquired infections. Effective treatment and prevention strategies are urgently needed. We investigated the potential of the S. aureus surface protein iron surface determinant B (IsdB) as a prophylactic vaccine against S. aureus infection. IsdB is an iron-sequestering protein that is conserved in diverse S. aureus clinical isolates, both methicillin resistant and methicillin sensitive, and it is expressed on the surface of all isolates tested. The vaccine was highly immunogenic in mice when it was formulated with amorphous aluminum hydroxyphosphate sulfate adjuvant, and the resulting antibody responses were associated with reproducible and significant protection in animal models of infection. The specificity of the protective immune responses in mice was demonstrated by using an S. aureus strain deficient for IsdB and HarA, a protein with a high level of identity to IsdB. We also demonstrated that IsdB is highly immunogenic in rhesus macaques, inducing a more-than-fivefold increase in antibody titers after a single immunization. Based on the data presented here, IsdB has excellent prospects for use as a vaccine against S. aureus disease in humans.
PMCID: PMC1418914  PMID: 16552052
21.  Infectious Virions Produced from a Human Papillomavirus Type 18/16 Genomic DNA Chimera 
Journal of Virology  2002;76(10):4723-4733.
The organotypic raft culture system has allowed the study of the differentiation-dependent aspects of the human papillomavirus (HPV) life cycle. However, genetic strategies to more completely understand the HPV life cycle are limited. The generation of chimeric viruses has been a useful tool in other virus systems to analyze infection and replication. To investigate the specificity of the interaction of nonstructural genes of one HPV type with the structural genes of another HPV type, we have replaced the L2 and L1 open reading frames (ORFs) of HPV type 18 (HPV18) with the L2 and L1 ORFs of HPV type 16 (HPV16). The resulting HPV18/16 chimeric construct was introduced into primary keratinocytes, where it was stably maintained episomally at a range of 50 to 100 copies of HPV genomic DNA, similar to that typically found in HPV-infected cells in vivo. The integrity of the HPV18/16 genomic DNA chimera was demonstrated. Upon differentiation in raft cultures, late viral functions, including viral DNA amplification, capsid gene expression, and virion morphogenesis, occurred. Chimeric HPV18/16 virions were purified from the raft cultures and were capable of infecting keratinocytes in vitro. Additionally, infection was specifically neutralized with human HPV16 virus-like particle (VLP)-specific antiserum and not with human HPV18 VLP-specific antiserum. Our data demonstrate that the nonstructural genes of HPV18 functionally interact with the structural genes of HPV16, allowing the complete HPV life cycle to occur. We believe that this is the first report of the propagation of chimeric HPV by normal life cycle pathways.
PMCID: PMC136126  PMID: 11967289
22.  Detection of Multiple Human Papillomavirus Types in Condylomata Acuminata Lesions from Otherwise Healthy and Immunosuppressed Patients 
Journal of Clinical Microbiology  1999;37(10):3316-3322.
Condylomata acuminata, or genital warts, are proliferative lesions of genital epithelium caused by human papillomavirus (HPV) infection. HPV types 6 and 11 are most often detected in these lesions. Genital lesions consistent with exophytic condylomata acuminata were removed by excision biopsy from 65 patients, 41 of whom were otherwise healthy individuals (control group) and 24 of whom had conditions known to cause immunosuppression. Histologically, the majority of the lesions were typical condylomata acuminata. Three lesions removed from immunosuppressed individuals also contained foci of moderate to severe dysplasia (intraepithelial neoplasia grade II/III). A recently developed PCR and reverse blot strip assay was used to determine the specific HPV types present in the genital lesions. With a set of oligonucleotide primers based on the same primer binding regions used for the MY09 and MY11 primer pair, this PCR assay detects the presence of 27 HPV types known to infect the genital tract. All but two condylomata acuminata contained either HPV type 6 or 11. The predominant type in the lesions from control patients was HPV 6, while lesions from immunosuppressed types most often contained HPV 11. Condylomata acuminata from immunosuppressed patients contained significantly more overall HPV types than lesions from the control group. HPV types associated with an increased risk of dysplasia (high-risk types) were detected in 42 (64.6%) of the total of 65 specimens; 18 (43.9%) specimens were detected in the 41 otherwise healthy individuals, and 24 (100%) specimens were detected in the 24 immunosuppressed patients. HPV 16 was the most common high-risk type detected, found in 21 of 65 (32.3%) specimens. After HPV types 6 and 11, HPV types 53 and 54 were the most frequently detected low-risk HPV types. This study demonstrates that a high percentage of condylomata acuminata lesions contain multiple HPV types, including types associated with a high risk of dysplastic abnormalities. Further studies are needed to determine the influence these additional HPV types have on the epidemiology of genital tract HPV infections and the natural history of condylomata acuminata, especially in immunosuppressed patients.
PMCID: PMC85555  PMID: 10488198
23.  Coiled-Coil Trigger Motifs in the 1B and 2B Rod Domain Segments Are Required for the Stability of Keratin Intermediate Filaments 
Molecular Biology of the Cell  2000;11(10):3539-3558.
Many α-helical proteins that form two-chain coiled coils possess a 13-residue trigger motif that seems to be required for the stability of the coiled coil. However, as currently defined, the motif is absent from intermediate filament (IF) protein chains, which nevertheless form segmented two-chain coiled coils. In the present work, we have searched for and identified two regions in IF chains that are essential for the stability necessary for the formation of coiled-coil molecules and thus may function as trigger motifs. We made a series of point substitutions with the keratin 5/keratin 14 IF system. Combinations of the wild-type and mutant chains were assembled in vitro and in vivo, and the stabilities of two-chain (one-molecule) and two-molecule assemblies were examined with use of a urea disassembly assay. Our new data document that there is a region located between residues 100 and 113 of the 2B rod domain segment that is absolutely required for molecular stability and IF assembly. This potential trigger motif differs slightly from the consensus in having an Asp residue at position 4 (instead of a Glu) and a Thr residue at position 9 (instead of a charged residue), but there is an absolute requirement for a Glu residue at position 6. Because these 13 residues are highly conserved, it seems possible that this motif functions in all IF chains. Likewise, by testing keratin IF with substitutions in both chains, we identified a second potential trigger motif between residues 79 and 91 of the 1B rod domain segment, which may also be conserved in all IF chains. However, we were unable to find a trigger motif in the 1A rod domain segment. In addition, many other point substitutions had little detectable effect on IF assembly, except for the conserved Lys-23 residue of the 2B rod domain segment. Cross-linking and modeling studies revealed that Lys-23 may lie very close to Glu-106 when two molecules are aligned in the A22 mode. Thus, the Glu-106 residue may have a dual role in IF structure: it may participate in trigger formation to afford special stability to the two-chain coiled-coil molecule, and it may participate in stabilization of the two-molecule hierarchical stage of IF structure.
PMCID: PMC15012  PMID: 11029054

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