• Co-administration of DNA encoding co-stimulatory molecules enhances the immune response to foreign antigen.
• Human metapneumovirus associated respiratory tract infection and disease; a CTL-based vaccine.
• Hepatitis B surface antigen (HBsAg) vector delivers protective CTL responses to disease-relevant foreign epitopes.
• Hepatitis B surface antigen-based vaccines for human neoplastic and infectious diseases.

Co-administration of DNA encoding co-stimulatory molecules enhances the immune response to foreign antigen.
Karen Herd, Carsten Wiethe, Robert Tindle.
Data from many sources indicate that immunological outcome depends on the milieu in which a T cell encounters antigen. Initiation of T cell immunity depends on the antigen presenting capacity of mature dendritic cells (DCs). After antigen capture DCs mature in response to inflammatory stimuli characterised by up-regulation of MHC and costimulatory molecules. DC-induced stimulation of CD8+ cytotoxic T lymphocytes (CTLs) directed to foreign epitopes is a vital component of the immune response to diseases which depend on CTL responses for disease prevention and control.
DCs process antigens to derive epitopes which localise at the cell surface in the cleft of the major histocompatibility (MHC) complex. The MHC/epitope complex is recognised by cognate T cell receptors and formation of this synapse signals T cell activation. This event requires a ‘second signal’, also known as a costimulatory signal, which is classically provided by ligation of CD28 on T cells with CD80 (B7-1) or CD86 (B7-2) on DCs, but is now known to be provided by a family of B7 molecules, by the tumour necrosis factor (TNF) superfamily, and by cytokines . The absence of second signalling may result in the DC transmitting a tolerogenic rather than an activation signal to the T cell.
In two recent Journal of Immunology papers ( Wiethe et al J.Immunol 170, 2912, 2003; Wiethe et al, J.Immunol 171,4121, 2003) we have shown that ex vivo manipulation of DCs to co-express certain costimulatory molecules (4-1BBL, RANK and RANKL), and a foreign antigen, elicits enhanced CTL responses to the foreign antigen when the DCs were used to immunise mice. However, a drawback of using ex vivo modified DC vaccines is that a separate vaccine must be made for each recipient and they are extremely labour intensive. In the present proposal we plan to develop a DNA-based vaccine approach which provides T cell and DC co-stimulation for enhanced vaccine efficacy.
The HYPOTHESIS to be tested is that co-immunisation with DNA encoding T cell and DC costimulatory molecules along with tumour antigen, will enhance effector and memory cytotoxic T cell responses and will give enhanced antigen-directed protection against disease.
The hypothesis will be tested in murine models, established in our laboratory, of Human papillomavirus (HPV16)-associated squamous carcinoma, in which the HPV16 E7 oncoprotein functions as a tumour specific antigen.
Overview of proposed research:

• We have generated plasmids containing E7 and costimulatory molecules RANK, RANKL, 4-1BB, 4-1BBL, and CD40, CD40L.
• Groups of mice have been immunized with E7 plasmid DNA with or without co-immunisation with plasmid DNA expressing costimulatory molecule receptors/ligand pairs. Immune response has been measured by in vitro correlates of CTL activation.
• Maximally effective combinations of E7 and costimulatory molecules will be selected for inclusion in pre-clinical trials of prophylactic and therapeutic approaches to tumour control.

If successful, an OUTCOME will be a simple generic vaccine strategy comparable to best-practice ‘boutique’ DCs vaccines applicable to those diseases for which disease-associated antigens are known ( ie. some cancers and infectious diseases) and which depend on a CTL response for their resolution.

Back

Human metapneumovirus associated respiratory tract infection and disease; a CTL-based vaccine.
Karen Herd, Huayang Guo, Suresh Mahalingam, Theo- Sloots, Michael Nissen, Robert Tindle.
Human metapneumovirus (hMPV) is proving to be a global and significant but hitherto unrecognized respiratory pathogen, for which a vaccine is highly desirable. The characteristics and clinical manifestations of (hMPV) closely resemble those of human respiratory syncytial virus (hRSV). Using established methodologies in our laboratory, we propose to develop cytotoxic T lymphocyte (CTL) DNA polytope vaccines for hMPV. The validity of the approach will be tested in ‘humanized’ murine models of hMPV infection. The approach will be extended to develop a combined vaccine for protection against both hRSV and hMPV.
The extent of hMPV related disease world wide only now becoming apparent. Comparison is invited with hRSV which causes 1 million deaths worldwide , and 2-4,000 paediatric hospitalizations in Australia, annually. We have on-going studies on the epidemiology of hMPV in Australian infants funded by NHMRC (Project grant #243702). (Nissen et al Med J Aust. 176 188 2002: Mackay et al . J.Clin.Microbiol. 41 100 2003).
Infection with respiratory pathogens begs the development of appropriate vaccines. Control of viral respiratory tract infection by the cytotoxic T lymphocyte (CTL) arm of the immune response is to be expected by analogy with other acute viral infections. In on-going studies we have defined CTL epitopes encoded by hMPV.
Specifically, the AIMS of the present proposal are:

1. To construct human and mouse DNA polytope vaccines encoding multiple hMPV CTL epitopes restricted through a number of MHC class 1 haplotypes, and to demonstrate that a) the hMPV human polytope will recall hMPV CTL responses restricted through multiple HLA class 1 loci in hMPV patients, and b) immunization of mice with the hMPV mouse polytope will elicit multiple hMPV-specific CTL responses.
2. To demonstrate that immunization with the hMPV mouse polytope vaccine will protect against a) new hMPV infection, and b) established hMPV infection and disease in a murine model of hMPV-associated respiratory disease.
3. To combine the above newly-acquired knowledge and ‘performance’ of human hMPV CTL epitopes with known RSV CTL epitopes from the literature to derive a combined hMPV/hRSV polytope vaccine whose function will be simultaneously protective for both hMPV and hRSV infection and disease.

The HYPOTHESES to be tested are:

• hMPV encodes immunogenic CTL epitopes restricted through a number of MHC class 1 haplotypes which may be incorporated into a DNA polytope vaccine, which will protect a MHC polymorphic (outbred) population against hMPV infection and associated respiratory tract disease.
• A CTL polyepitope vaccine comprising CTL epitopes of hMPV and hRSV will concomitantly protect against infection and disease caused by both these pathogens.

The NOVEL FEATURES and potential OUTCOME of this proposal are:

Hepatitis B surface antigen (HBsAg) vector delivers protective CTL responses to disease-relevant foreign epitopes.
Huayang Guo, Michael Mather, Kristy Edgtton, Robert Tindle
Most infectious diseases and some cancers are preventable by appropriate vaccination. What is lacking is a way to deliver the vaccines to elicit the appropriate immune response(s) to prevent infection or cure disease. We propose a systematic approach to the development of hepatitis B small envelope protein (hepatitis B surface antigen; (HBsAg)) as an adaptable vaccine vector for the delivery of multiple protective epitopes against a range of diseases. Ca 100 HBsAg molecules assemble with host-derived lipids into empty envelope virus-like particles (VLPs) without the participation of nucleocapsids. HBsAg administered as VLP- or as DNA-vaccines induce potent HBsAg-directed immune responses due to their particulate nature and repeated sub-unit structure. Indeed HBsAg is the pan-globally licensed human vaccine for hepatitis B infection. Attempts by other workers to exploit the outstanding immunogenicity of HBsAg as a vector modality for the delivey of foreign CTL epitopes have largely failed, due to insertions compromising HBsAg structural/functional integrity. Using a novel strategy developed in our laboratory of deletion of endogenous CTL epitopes, and their replacement with inserted foreign CTL epitopes, we have largely overcome these difficulties. We propose to exploit HBsAg as a powerful vaccine vector to deliver multiple (we estimate up to 10) inserted tumor- and pathogen- CTL epitopes restricted through a selection of major histocompatability (MHC) class 1 alleles. We will determine the efficacy of the recombinant HBsAg vaccines in transgenic murine models of human neoplastic and/or infectious disease established in our laboratories. We already have preliminary data (with human papillomavirus and respiratory syncytial virus) to demonstrate the generation of multiple effector and memory CTL responses, and simultaneous protection against several diseases by immunization with a single recombinant HBsAg encoding multiple different protective epitopes.

.

The HYPOTHESES to be tested are:

• CTL epitopes of infectious agents or tumors can be inserted into multiple locations in HBsAg and the resulting rVLPs retain integral tertiary structure and the capacity for secretion.
• Recombinant (r-)HBsAg vaccine administered as VLPs or as DNA will elicit long-lived effector and memory CTL responses directed to multiple inserted epitopes restricted through multiple class 1 haplotypes.
• rHBsAg vaccination will invoke effective prophylactic and therapeutic responses against challenge with infectious agents and tumors in murine models of human disease.
•  rHBsAg may be developed which encodes both foreign CTL and B cell epitopes of a pathogen, that may be applied as single vaccine to induce simultaneous CTL and antibody responses which are synergistic for protection against infection.

The NOVEL FEATURES of this proposal are a novel generic recombinant vaccine strategy potentially applicable to all pathogens and tumours for which protective epitopes are/will be known, based on an already licensed vaccine moiety (HBsAg), and a novel technology to generate functionally intact recombinant HBsAg molecules.
The OUTCOME will be an aptable multivalent vaccine technology, applicable to all infectious diseases and cancers for which protective epitopes are/will be known, and which depend on appropriate immune responses for their resolution.

Back

Hepatitis B surface antigen-based vaccines for human neoplastic and infectious diseases.
Oscar Haigh, Jacqeline Kattenbelt, Allan Gould, Robert Tindle.
Specifically, rHBsAg vaccines recombinant for tumour-associated antigens of human papillomavirus will be developed. As time allows, a further vaccine recombinant for avian influenza virus (AIV H5N1) will be developed.
The proposal is based on existing expertise in the Viral Immunology Unit (VIU) at the Clinical Medical Virology Centre a) in the construction and evaluation of HBsAg-based recombinant vectors for the delivery of protective immune responses , and b) in vaccines for HPV-associated squamous cancer.
The project will develop HBsAg–HPV and –AIV vaccine(s) which will be tested in murine models of immune response induction and protection. The project may lead to commercial development. The HBsAg-AIV vaccine will interface with collaborative studies between the Viral Immunology Unit and CSIRO (Geelong).
The SPECIFIC AIMS of the proposal are:

• To derive DNA constructs encoding HBsAg recombinant for E6 and E7 oncoproteins of HPV 16, and from these constructs, to derive r-HBsAg virus-like particles (VLPs). Further constructs recombinant for the H5 surface glycoprotein of AIV will be undertaken.
• To demonstrate that immunization of mice with:

  • a) HBsAg-HPV DNA- or VLP- vaccines elicits E6- and E7-directed cytotoxic T-lymphocyte (CTL) responses which kill E6- and E7 expressing tumour cells in vitro, and prevent the establishment and growth of HPV-associated tumour in ‘humanised’ HLA transgenic mouse models.
  • b) HBsAg-AIV vaccines elicit H5-directed neutralizing antibody and CTL responses which protect against H5N1 infection in a murine model of AIV disease

The HYPOTHESES to be tested are that the HBsAg vaccines recombinant for immunogenic proteins of HPV ( or AIV), administered as DNA or VLP vaccines will:

• elicit HPV (or AIV) directed cellular and humoral responses
• protect against HPV-associated neoplastic disease (or AIV-associated respiratory disease).

The NOVEL FEATURES of the proposal are the exploitation of nascent HBsAg vector technology to develop superior vaccines for neoplastic and infectious diseases relevant to humans.
The OUTCOME the specific outcome will be a potential therapeutic vaccine for cervical carcinoma, and (as time allows) a prophylactic and therapeutic vaccine for AIV infection in humans.

Back