HUMAN PAPILLOMA VIRUS

• Therapeutic Vaccines
• Prophylactic Vaccines
• Development of a Mouse Model for Efficacy Testing of Vaccines Against a Genital
  Infection with HPV-16
• Human Immunodeficiency Virus
• Rabies Virus
• Tumor Vaccines to p53
• Vaccines to Brain Tumors
• Avenues to Circumvent Immune Responses Against Therapeutic Proteins Delivered
  During Gene Therapy
  

Therapeutic vaccines (A. Wlazlo, Ph.D.)

Certain types of human papillomaviruses most notably types 16 and 18 are causative for cervical cancer, the second most common cause of cancer morbidity and mortality in women worldwide. Two of the viral early proteins, i.e., the E6 and E7 proteins dysregulate cell cycle control by inactivating the p53 and retinoblastoma gene products respectively. Cervical cancer cells express these two viral oncoproteins which thus provide suitable target antigens for active immunotherapy. The laboratory developed a set of different vaccines based on viral recombinants or DNA vaccines. These vaccines express either wild-type E6 or E7 or fusion proteins thereof designed to facilitate targeting of the proteins towards the MHC class I or II processing pathway for optimized induction of CD4 or CD8 mediated immune responses. The efficacy of these vaccines is being assessed in mouse tumor models. One of the vaccines, i.e., the E1-deleted adenoviral recombinant to E7 was shown to provide near complete protection through CD8+ T cell mediated immune effector mechanisms which are currently being analyzed in more detail.

HPV-16 is a sexually transmitted infectious agent that infects more than 20% of sexually active women in the US thus putting them at risk for the eventual development of cervical cancer. A prophylactic vaccine, designed to prevent HPV infection, should induce both systemic and mucosal neutralizing antibodies to the major capsid protein (L1) of HPV-16. Experimental vaccines based on virus like particles composed of the L1 protein are currently undergoing large-scale clinical trials. The laboratory developed an alternative vaccine regimen based on a DNA vaccine used for priming and an E1-deleted adenoviral recombinant for intranasal booster immunization both expressing the L1 of HPV-16. This vaccine regimen induces a vaginal antibody response to conformational epitopes of the L1 of HPV-16. Further avenues to augment the magnitude and duration of the vaginal antibody response are being explored.

It is indispensable to test the efficacy of vaccines against HPV-16 to induce immune effector mechanisms at the mucosal surfaces. This project focuses on the development of a mouse model that allows testing the efficacy of vaccines developed in our laboratory against genital infection with HPV-16. As HPV-16 does not infect mice, an attenuated rabies virus that readily infects the mouse mucosa will be genetically modified to express the E6 and E7 oncoproteins of HPV-16. These constructs will be used to vaginally infect naïve and vaccinated mice. The degree of protection against infection with these constructs will be determined indicating the efficiency of the vaccines. The local cellular immune response will be analyzed and the effect of different vaccination protocols will be compared. The information derived from these experiments is expected to lead to a better understanding of the cellular immune response to infections of the genital tract and of the effect of vaccination on mucosal immunity.

A prophylactic vaccine to HIV-1 has to induce potent cellular and humoral immune responses to antigens of HIV-1. HIV-1 is in most cases sexually transmitted and effective prevention of a systemic chronic infection is assumed to require antibodies and memory T cells at the mucosal sites of viral entry. Pursuant to these goals, the laboratory has developed a vaccine regimen in a rabies mouse model, which optimally induces mucosal antibody secretion and systemic cytolytic T cell responses. Priming with a DNA vaccine against a model (rabies) antigen, followed by booster immunization with an intranasally applied, E1-deleted adenoviral recombinant expressing the antigen of interest, induces potent serous antibody responses, as well as a strong, prolonged antibody response at the vaginal mucosa of immunized mice. DNA vaccine priming also reduced the subsequent antibody response to the carrier adenoviral antigens. Further, in mice pre-immune to adenovirus, DNA vaccine priming overcomes the impaired B cell response to rabies antigen expressed by the adenoviral recombinant vaccine. The vaginal B cell response can further be enhanced by interleukin (IL)-4, IL-10, or IL-5, co-administered as genetic adjuvants with DNA vaccine, prior to subsequent booster immunization with i.n. recombinant adenoviral vaccine.

This type of a vaccine regimen is now being applied to the far more complex HIV system by generating vaccines expressing the env or gag of HIV-1 or SHIV, a chimeric virus of HIV and simian immunodeficiency virus. Plasmid DNA vaccines are being developed as reagents for 'priming' and recombinant adenoviral vaccine for subsequent 'booster' immunizations.

Over the last 12 years the laboratory has analyzed in depth immune responses to different antigens of rabies virus, a negative stranded DNA virus that is endemic in carnivores in most countries. The results of these studies formed the basis for the development of vaccines to rabies virus that upon passing pre-clinical rodent efficacy tests are now in part being tested in larger mammals.

The rabies virus model by its virtue of being a well-characterized and simple model serves the entire group to establish basic immunological principles pertinent for vaccine development. Once such principles have been established the gained knowledge is the transferred to the far complex HIV and HPV vaccine systems.

The rabies system was used to establish that DNA vaccines could induce long-lasting protective antibody responses against inrtracerebral challenge with a potentially lethal virus. This model was also used to show that vectors encoding cytokines could modulate the immune response to DNA vaccines thus establishing the principle of genetic adjuvants.

The rabies virus model served to demonstrate that intranasal immunization with E1-deleted adenoviral recombinant induces a strong genital antibody response, which is expected to be paramount for the prevention of sexually transmitted infections.

The laboratory currently uses this model to test the effect of endogenous cytokines on the transcriptional regulation of DNA vaccine encoded antigens.

The efficacy of tumor vaccines expressing the p53 oncoprotein is being assessed in mouse models. The p53 protein is an attractive target for immunotherapy, since overexpression of mutated or wild-type p53 protein are common genetic alterations found in a variety of human tumors. A vaccinia virus recombinant vaccine expressing wild-type mouse p53 (p53wt), mounts a p53-specific immune response that provides partial protection against subsequent challenge with murine tumor cell lines expressing high levels of p53. Protection is mediated by a complex immune response composed of cytolytic T lymphocytes and CD4+ T helper cells, in addition to natural killer cells. The efficacy of the vaccine is significantly improved by using recombinant mouse interleukin (rIL)-12 as an adjuvant following tumor challenge. A second prototype vaccine is based on plasmid vectors, so-called DNA which express the mouse p53 protein as a fusion protein linked to sequences that modify its cellular localization and thus influence the antigen processing pathway.

Further studies are in progress to define the immune effector mechanisms against p53 and to elucidate which p53 abnormalities render this self-protein suitable for a tumor rejection antigen.

The aim of these studies is to study the efficacy of dendritic cell based vaccines against tumors in the central nervous system (CNS) in a mouse glioblastoma model. Immunization of mice with ex vivo expanded dendritic cells pulsed with RNA from glioblastoma cells results in protection of mice against intracerebral challenge with the homologous tumor cells, Vaccine efficacy is improved by co-administration of interleukin (rIL)-12. The immune mechanism underlying the vaccine induced protection against an intracranially (i.c.) progressing tumor are being investigated.

Although the laboratory otherwise focuses on the induction of immune responses against harmful agents one project aims at the opposite that is the induction of immunological tolerance against a therapeutic protein. The general applicability of gene therapy depends on the efficient transfer and long term expression of an exogenously induced gene in a specific tissue. Nevertheless, many vectors and transgenes elicit potent innate and antigen specific B and T-cell immune responses that ultimately cause the elimination of the transgene and cells that express it. The laboratory is investigating avenues to circumvent responses within the realm of gene therapy.