Mucosal but not parenteral immunization with purified human papillomavirus type 16 virus-like particles induces neutralizing titers of antibodies throughout the estrous cycle of mice.

We have recently shown that nasal immunization of anesthetized mice with human papillomavirus type 16 (HPV16) virus-like particles (VLPs) is highly effective at inducing both neutralizing immunoglobulin A (IgA) and IgG in genital secretions, while parenteral immunization induced only neutralizing IgG. Our data also demonstrated that both isotypes are similarly neutralizing according to an in vitro pseudotyped neutralization assay. However, it is known that various amounts of IgA and IgG are produced in genital secretions along the estrous cycle. Therefore, we have investigated how this variation influences the amount of HPV16 neutralizing antibodies induced after immunization with VLPs. We have compared parenteral and nasal protocols of vaccination with daily samplings of genital secretions of mice. Enzyme-linked immunosorbent assay analysis showed that total IgA and IgG inversely varied along the estrous cycle, with the largest amounts of IgA in proestrus-estrus and the largest amount of IgG in diestrus. This resulted in HPV16 neutralizing titers of IgG only being achieved during diestrus upon parenteral immunization. In contrast, nasal vaccination induced neutralizing titers of IgA plus IgG throughout the estrous cycle, as confirmed by in vitro pseudotyped neutralization assays. Our data suggest that mucosal immunization might be more efficient than parenteral immunization at inducing continuous protection of the female genital tract.

International collaborative proficiency study of Human Papillomavirus type 16 serology.

We performed an international proficiency study of Human Papillomavirus (HPV) type 16 serology. A common methodology for serology based on virus-like particle (VLP) ELISA was used by 10 laboratories in 6 continents. The laboratories used the same VLP reference reagent, which was selected as the most stable, sensitive and specific VLP preparation out of VLPs donated from 5 different sources. A blinded proficiency panel consisting of 52 serum samples from women with PCR-verified HPV 16-infection, 11 control serum samples from virginal women and the WHO HPV 16 International Standard (IS) serum were distributed. The mean plus 3 standard deviations of the negative control serum samples was the most generally useful "cut-off" criterion for distinguishing positive and negative samples. Using sensitivity of at least 50% and a specificity of 100% as proficiency criteria, 6/10 laboratories were proficient. In conclusion, an international Standard Operating Procedure for HPV serology, an international reporting system in International Units (IU) and a common "cut-off" criterion have been evaluated in an international HPV serology proficiency study.

Functions of the human papillomavirus type 16 E4 protein

1. Abstract Cervical cancer is thought to be the consequence of infection by human papillomaviruses (HPV). In the majority of cases, DNA from HPV type 16 (HPV16) is found in malignant cervical lesions. The initial steps leading to transformation of an infected cell are not clearly understood but in most cases, disruption and integration of the episomal viral DNA must take place. As a consequence, the E2 and E4 genes are usually not expressed whereas the E6 and E7 oncogenes are highly expressed. However, in a normal infection in which the viral DNA is maintained as an episome, all viral genes are expressed. The pattern according to which the viral proteins are made, and therefore the life cycle of the virus, is tightly linked to the differentiation process of the host keratinocyte. The study of the viral oncogenes E6 and E7 has revealed crucial functions in the process of malignant transformation such as degradation of the p53 tumor suppressor protein, deregulation of the Retinoblastoma protein pathway and activation of the telomerase ribonucleoprotein. All these steps are necessary for cancerous lesions to develop. However, the loss of the E2 gene product seems to be necessary for sufficient expression of E6 and E7 in order to achieve such effects. In normal infections, the E4 protein is made abundantly in the later stages of the viral life cycle. Though extensive amounts of work have been carried out to define the function of E4, it still remains unclear. In this study, several approaches have been used to try and determine the functions of E4. First, a cell-penetrating fusion protein was designed and produced in order to circumvent the chronic difficulties of expressing E4 in mammalian cells. Unfortunately, this approach was not successful due to precipitation of the purified fusion protein. Second, the observation that E4 accumulates in cells having modified their adhesion properties led to the hypothesis that E4 might be involved in the differentiation process of keratinocytes. Preliminary results suggest that E4 triggers differentiation. Last, as E4 has been reported to collapse the cytokeratin network of keratinocytes, a direct approach using atomic force microscopy has allowed us to test the potential modification of mechanical properties of cells harboring reorganized cytokeratin networks. If so, a potential role for E4 in viral particle release could be hypothesized. 2. Résumé Il a été établi que le cancer du col de l'utérus se développe essentiellement à la suite d'une infection par le virus du papillome humain (HPV). Dans la majorité des cas analysés, de l'ADN du HPV de type 16 (HPV16) est détecté. Les étapes initiales de la transformation d'une cellule infectée sont mal connues mais il semble qu'une rupture du génome viral, normalement épisomal, suivi d'une intégration dans le génome de la cellule hôte soient des étapes nécessaires dans la plupart des cas. Or il semble qu'il y ait une sélection pour les cas où l'expression des oncogènes viraux E6 et E7 soit favorisée alors que l'expression des gènes E2 et E4 est en général impossible. Par contre, dans une infection dite normale où le génome viral n'est pas rompu, il n'y pas développement de cancer et tous les gènes viraux sont exprimés. L'ordre dans lequel les protéines virales sont produites, et donc le cycle de réplication du virus, est intimement lié au processus de différentiation de la cellule hôte. L'étude des protéines oncogènes E6 et E7 a révélé des fonctions clés dans le processus de transformation des cellules infectées telles que la dégradation du suppresseur de tumeur p53, la dérégulation de la voie de signalisation Rb ainsi que l'activation de la télomérase. Toutes ces activités sont nécessaires au développement de lésions cancéreuses. Toutefois, il semble que l'expression du gène E2 doit être empêchée afin que suffisamment des protéines E6 et E7 soient produites. Lorsque le gène E2 est exprimé, et donc lorsque le génome viral n'est pas rompu, les protéines E6 et E7 n'entraînent pas de telles conséquences. Le gène E4, qui se trouve dans la séquence codante de E2, a aussi besoin d'un génome viral intact pour être exprimé. Dans une infection normale, le gène E4 est exprimé abondamment dans les dernières étapes de la réplication du virus. Bien que de nombreuses études aient été menées afin de déterminer la fonction virale à E4, aucun résultat n'apparaît évident. Dans ce travail, plusieurs approches ont été utilisées afin d'adresser cette question. Premièrement, une protéine de fusion TAT-E4 a été produite et purifiée. Cette protéine, pouvant entrer dans les cellules vivantes par diffusion au travers de la membrane plasmique, aurait permis d'éviter ainsi les problèmes chroniques rencontrés lors de l'expression de E4 dans les cellules mammifères. Malheureusement, cette stratégie n'a pas pu être utilisée à cause de la précipitation de la protéine purifiée. Ensuite, l'observation que E4 s'accumule dans les cellules ayant modifié leurs propriétés d'adhésion a suggéré que E4 pourrait être impliqué dans le procédé de différentiation des kératinocytes. Des résultats préliminaires supportent cette possibilité. Enfin, il a été montré que E4 pouvait induire une réorganisation du réseau des cytokératines. Une approche directe utilisant le microscope à force atomique nous a ainsi permis de tester une potentielle modification des propriétés mécaniques de cellules ayant modifié leur réseau de cytokératines en présence de E4. Si tel est le cas, un rôle dans la libération de particules virales peut être proposé pour E4.

Enhanced anti-tumor effect of a gene gun-delivered DNA vaccine encoding the human papillomavirus type 16 oncoproteins genetically fused to the herpes simplex virus glycoprotein D

Anti-cancer DNA vaccines have attracted growing interest as a simple and non-invasive method for both the treatment and prevention of tumors induced by human papillomaviruses. Nonetheless, the low immunogenicity of parenterally administered vaccines, particularly regarding the activation of cytotoxic CD8+ T cell responses, suggests that further improvements in both vaccine composition and administration routes are still required. In the present study, we report the immune responses and anti-tumor effects of a DNA vaccine (pgD-E7E6E5) expressing three proteins (E7, E6, and E5) of the human papillomavirus type 16 genetically fused to the glycoprotein D of the human herpes simplex virus type 1, which was administered to mice by the intradermal (id) route using a gene gun. A single id dose of pgD-E7E6E5 (2 µg/dose) induced a strong activation of E7-specific interferon-γ (INF-γ)-producing CD8+ T cells and full prophylactic anti-tumor effects in the vaccinated mice. Three vaccine doses inhibited tumor growth in 70% of the mice with established tumors. In addition, a single vaccine dose consisting of the co-administration of pgD-E7E6E5 and the vector encoding interleukin-12 or granulocyte-macrophage colony-stimulating factor further enhanced the therapeutic anti-tumor effects and conferred protection to 60 and 50% of the vaccinated mice, respectively. In conclusion, id administration of pgD-E7E6E5 significantly enhanced the immunogenicity and anti-tumor effects of the DNA vaccine, representing a promising administration route for future clinical trials.

Charge virale intégrée du papillomavirus de type 16 dans la maladie anale préinvasive

L’histoire naturelle de l’infection anale par le virus du papillome de type 16 (VPH-16) est mal définie pour les hommes ayant des relations sexuelles avec d’autres hommes (HARSAHs) VIH-séropositifs. Le but de cette étude était d’évaluer l’association entre la charge épisomale et intégrée du VPH-16 et la progression de la néoplasie intraépithéliale anale (AIN). Les charges épisomales et intégrées du VPH-16 furent mesurées par PCR quantitatif en temps réel sur 665 spécimens anaux obtenus de 135 hommes VPH-16-positifs participant à l’étude prospective HIPVIRG (Human Immunodeficiency and Papilloma VIrus Research Group). Le grade de l’AIN fut déterminé sur des biopsies obtenues lors des anuscopies à haute résolution périodiques. L’intégration du VPH-16 fut confirmée par DIPS-PCR pour démontrer la présence de jonctions virales-cellulaires. La charge épisomale du VPH-16 [ratio de cote (OR) 1.5, intervalle de confiance (IC) à 95%=1.1–2.1], le nombre de types de VPH [OR 1.4 (IC 95%=1.1–1.8)] et le tabagisme actuel [OR 4.8 (IC 95%=1.3–18.6)], mais non la charge intégrée, furent associés aux lésions de haut-grade (AIN-2,3) après ajustement pour l’âge et le décompte des lymphocytes CD4. La charge épisomale du VPH-16 était le seul facteur prédictif de progression de l’AIN de bas-grade (AIN-1) vers l’AIN-2,3 [OR 8.0 (IC 95%=1.2–55.4)]. Les spécimens avec une charge épisomale du VPH-16 élevée étaient moins susceptibles de contenir de l’intégration [OR 0.5 (IC 95%=0.3–0.8)]. L’intégration du VPH-16 fut détectée en absence d’AIN, dans l’AIN-1 et dans l’AIN-2,3. L’analyse des jonctions virales-cellulaires ne permit pas d’identifier un site d’intégration spécifique.

Immune Responses and Therapeutic Antitumor Effects of an Experimental DNA Vaccine Encoding Human Papillomavirus Type 16 Oncoproteins Genetically Fused to Herpesvirus Glycoprotein D

Recombinant adenovirus or DNA vaccines encoding herpes simplex virus type 1 (HSV-1) glycoprotein D (gD) genetically fused to human papillomavirus type 16 (HPV-16) oncoproteins (E5, E6, and E7) induce antigen-specific CD8(+) T-cell responses and confer preventive resistance to transplantable murine tumor cells (TC-1 cells). In the present report, we characterized some previously uncovered aspects concerning the induction of CD8(+) T-cell responses and the therapeutic anticancer effects achieved in C57BL/6 mice immunized with pgD-E7E6E5 previously challenged with TC-1 cells. Concerning the characterization of the immune responses elicited in mice vaccinated with pgD-E7E6E5, we determined the effect of the CD4(+) T-cell requirement, longevity, and dose-dependent activation on the E7-specific CD8(+) T-cell responses. In addition, we determined the priming/boosting properties of pgD-E7E6E5 when used in combination with a recombinant serotype 68 adenovirus (AdC68) vector encoding the same chimeric antigen. Mice challenged with TC-1 cells and then immunized with three doses of pgD-E7E6E5 elicited CD8(+) T-cell responses, measured by intracellular gamma interferon (IFN-gamma) and CD107a accumulation, to the three HPV-16 oncoproteins and displayed in vivo antigen-specific cytolytic activity, as demonstrated with carboxyfluorescein diacetate succinimidyl ester (CFSE)-labeled target cells pulsed with oligopeptides corresponding to the H-2D(b)-restricted immunodominant epitopes of the E7, E6, or E5 oncoprotein. Up to 70% of the mice challenged with 5 x 10(5) TC-1 cells and immunized with pgD-E7E6E5 controlled tumor development even after 3 days of tumor cell challenge. In addition, coadministration of pgD-E7E6E5 with DNA vectors encoding pGM-CSF or interleukin-12 (IL-12) enhanced the therapeutic antitumor effects for all mice challenged with TC-1 cells. In conclusion, the present results expand our previous knowledge on the immune modulation properties of the pgD-E7E6E5 vector and demonstrate, for the first time, the strong antitumor effects of the DNA vaccine, raising promising perspectives regarding the development of immunotherapeutic reagents for the control of HPV-16-associated tumors.

Expression of human papillomavirus type 16 E7 oncoprotein alters keratinocytes expression profile in response to tumor necrosis factor-alpha

Acute expression of E7 oncogene from human papillomavirus (HPV) 16 or HPV18 is sufficient to overcome tumor necrosis factor (TNF)-alpha cytostatic effect on primary human keratinocytes. In the present study, we investigated the molecular basis of E7-induced TNF resistance through a comparative analysis of the effect of this cytokine on the proliferation and global gene expression of normal and E7-expressing keratinocytes. Using E7 functional mutants, we show that E7-induced TNF resistance correlates with its ability to mediate pRb degradation and cell transformation. On the other hand, this effect does not depend on E7 sequences required to override DNA damage-induced cell cycle arrest or extend keratinocyte life span. Furthermore, we identified a group of 66 genes whose expression pattern differs between normal and E7-expressing cells upon cytokine treatment. These genes are mainly involved in cell cycle regulation suggesting that their altered expression may contribute to sustained cell proliferation even in the presence of a cytostatic stimulus. Differential expression of TCN1 (transcobalamin I), IFI44 (Interferon-induced protein 44), HMGB2 (high-mobility group box 2) and FUS [Fusion (involved in t(12; 16) in malignant liposarcoma)] among other genes were further confirmed by western-blot and/or real-time polymerase chain reaction. Moreover, FUS upregulation was detected in HPV-positive cervical high-grade squamous intraepithelial lesions when compared with normal cervical tissue. Further evaluation of the role of such genes in TNF resistance and HPVassociated disease development is warranted.

Human papillomavirus type 16 variants in cervical cancer from an admixtured population in Brazil

Several studies indicate that molecular variants of HPV-16 have different geographic distribution and risk associated with persistent infection and development of high-grade cervical lesions. In the present study, the frequency of HPV-16 variants was determined in 81 biopsies from women with cervical intraepithelial neoplasia grade III or invasive cervical cancer from the city of Belem, Northern Brazil. Host DNAs were also genotyped in order to analyze the ethnicity-related distribution of these variants. Ninie different HPV-16 LCR variants belonging to four phylogenetic branches were identified. Among these, two new isolates were characterized. The most prevalent HPV-16 variant detected was the Asian-American B-2,followed by the European B-12 and the European prototype. Infections by multiple variants were observed in both invasive cervical cancer and cervical intraepithelial neoplasia grade III cases. The analysis of a specific polymorphism within the E6 viral gene was performed in a subset of 76 isolates. The E6-350G polymorphism was significantly more frequent in Asian-American variants. The HPV-16 variability detected followed the same pattern of the genetic ancestry observed in Northern Brazil, with European, Amerindian and African roots. Although African ancestry was higher among women infected by the prototype, no correlation between ethnical origin and HPV-16 variants was found. These results corroborate previous data showing a high frequency of Asian-American variants in cervical neoplasia among women with multiethnic origin.

Type 16 Design C Port Side; SS Chattahoochee

Print No: 66.; Initials Lower Right: ELW (Everett Longley Warner)

Type 16 Design C Starboard Side; SS Chattahoochee

Print No:80.; Initials Lower Right: ELW (Everett Longley Warner)

Type 16 Design B Port Side; SS Chattahoochee

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Type 16 Design B Starboard Side; SS Chattahoochee

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Type 16 Design D Port Side; SS Chattahoochee

Print No:73; ; Initials Lower Left: SN; Initials Lower Left: NH; Initials Lower Left: WR

Type 16 Design D Starboard Side; SS Chattahoochee

Print No:80; ; Initials Lower Left: SN; Initials Lower Left: NH; Initials Lower Left: WR

Type 16 Design A Port Side; SS Chattahoochee

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