Overview

A triumph of investigative life sciences in the last 70 years has been the unravelling of the complexities of the immune system. We now have a fair understanding of how the body has evolved to withstand the onslaught of microbial invaders who wish to use the body’s biochemistry and physiology to propagate their own genetic material, at the same time sometimes causing disease. The body also uses it’s immune system to protect itself from attack from within. In the very processes of tissue growth, repair and regeneration, aberrant cells arise, which (often with the help of outside environmental stimuli eg. UV irradiation, certain chemicals) grow out of control and give rise to tumours. By careful monitoring, the immune system can recognise these aberrant cells as ‘not right’ and, with the help of specialised ‘killer’ immune cells, remove them.
Vaccines work by stimulating the immune system to react against specific diseases. Testimony to the work of immunologists is that almost all the diseases that threaten young life are now preventable by vaccines given in childhood vaccination schedules.
Many challenges remain. We are still discovering the subtleties of the ways in which the immune system combats the invaders, and importantly, how it avoids attacking the body itself in the process. New immune functions and pathways are being discovered. But the invading microorganisms are not passive victims of immune onslaught. They in their turn have evolved mechanisms for avoiding the body’s immune attack, for example by producing chemicals to direct the body’s immune mechanisms in the wrong direction. And so a continual evolutionary cat-and-mouse game occurs in which the body’s immune system and the invading microorganism each tries to get the upper hand. Some invaders can over-ride the immune system and have resisted attempts to control them by vaccines – they are far too clever for the body and for the scientists. One doesn’t have to think of HIV/AIDS; even Respiratory Syncytial Virus (RSV), the cause of the majority of hospitalizations of children with respiratory infection, falls into this category. The work of the immunologist who studies basic immune mechanisms, and the vaccinologist who translates immune mechanisms into clinical practice, is never done.
We have a number of avenues of investigation currently underway in the Viral Immunology Unit. Dr Scott Thomson is interested in how the cells of the immune system fine-tune and optimise the response that they make by using specialised ‘costimulatory molecules’. Karen Herd had concluded a fine series of experiments in which she has definitively characterised the killer-cells (otherwise known as ‘CTL’) response made by mice and by human patients to human metapneumovirus (hMPV).
hMPV is a pathogen which causes respiratory disease particularly in young children, the elderly and the immunocompromised. In conjunction with collaborators at the University of Canberra, Karen has carefully plotted the immune course of infection with hMPV and has developed an experimental vaccine. Ph.D student Oscar Haigh has exploited an already existing human vaccine, hepatitis B surface antigen, as a framework on which to build a vaccine to treat cancers which are caused by human papillomavirus (HPV). Jason Cheong has investigated whether costimulatory molecules can be delivered by hepatitis B surface antigen.
These projects are among the successes. The very nature of research is that it doesn’t always yield the results one hopes for. Our work to base a vaccine to treat HPV-associated tumours on the HPV E5 molecule and our work exploiting Hepatitis delta antigen as a vaccine vehicle, have ceased because the results of quite extensive experimentation made it clear that these avenues were simply not the right way to go.
A collaboration with Associate Professor Nigel McMillan and his colleagues at Diamantina Institute, Princess Alexandra Hospital, has been particularly productive. It was shown by Dr Wenyi Gu, and by Dr Melanie Cochrane that treatment of tumour cells with a regulator molecule called RNAi enhanced the visibility of the tumour to the immune system, resulting in decreased tumour growth.

Research Projects

Hepatitis B surface Antigen-based Vaccine for Human Neoplastic Disease
Oscar Haigh, Allan Gould, Jacqueline Kattenbelt, Scott Thomson, Robert Tindle

Hepatitis B surface antigen (HBsAg) vaccines elicit strong cytotoxic T-lymphocyte (CTL) responses when delivered as DNA or virus-like particles. When foreign antigens are coexpressed with HBsAg as a fusion protein, powerful antigen-specific CTL responses are achieved. Therefore, HBsAg is an attractive vaccine vector for delivery of disease-related foreign epitopes. The capacity to elicit CTL responses compares favourably when rHBsAg vaccines are delivered by DNA rather than VLP-modalities. Human papillomavirus (HPV)-associated carcinoma is the leading cause of death in women worldwide. A major feature of the HPV lifecycle is the immortalisation and neoplastic growth of the squamous epithelium, a result of HPV E6 and E7 oncogene expression. E6 and E7 oncoproteins are essential to maintain a transformed cell phenotype and are present in all stages of cervical intra-epithelial neoplasia (CIN), which make them ideal targets for immunotherapeutic therapy.
In this study, we have generated an HBsAg-based vaccine platform, by splice overlap extension (SOE)-PCR, encoding the E6 and E7 oncoproteins fused to HBsAg (E7HBE6). E6 and E7 were mutated to eliminate potential oncogenicity and were codon optimised for enhanced expression. The effector and memory CTL response, as well as tumour protection and therapy afforded by immunisation with HBsAg-HPV vaccine was evaluated in mice. The HBsAg-HPV construct was evaluated for the potential to form VLPs.
Insect cells expressed HBsAg-HPV protein at low levels which appeared to form aggregates and did not demonstrate VLP formation. The formation of HBsAg-HPV VLPs and HBsAg-HPV protein were undetectable when expressed from two different mammalian cell lines. Observations within these experiments suggested that the HBsAg-HPV protein maybe unstable.
Studies were conducted using a DNA plasmid vectored HBsAg-HPV vaccine. We demonstrated that HBsAg-HPV DNA vaccine was immunogenic at a single low dose which was enhanced by boosting. Effector and memory CTL responses to E6 and E7 were maintained for 23 weeks post-immunisation. A maximal CTL response was achieved from a single dose at 30-100μg with a significant response also detected from a single dose of 10μg. When a dominant epitope was eliminated from within HBsAg-HPV, an immunodominance hierarchy was demonstrated. The presence of a subdominant epitope enhanced the response to the dominant epitope, at the cost of a decrease in its own effector CTL response. The immunodominance effect was decreased by separation of antigens and furthermore by administration of antigens at separate anatomical sites. Antigenic sin was demonstrated by the immunisation of HBsAg-wildtype-experienced mice with recombinant HBsAg-HPV DNA vaccines. Understanding the CTL response interplay between the two HPV antigens, within the context of the pE7HBE6 vaccine is important for its application as a tumour therapy.
Prevention of the growth of subcutaneous TC-1 tumour (HPV-16-associated) was achieved in 100% of mice immunised with HBsAg-HPV DNA vaccine, compared to mice immunised with HBsAg wildtype DNA vaccine, all of which presented with tumour. When used as a therapeutic agent, a significant increase in survival and decreased tumour volume were attained. In an intravenously challenged murine TC-1 metastasis model of therapy, mice immunised with HBsAg-HPV DNA vaccine remained 100% tumour free compared to mice immunised with HBsAg DNA, of which 80% displayed lung tumours. The results from this study demonstrate that a HBsAg-vectored DNA vaccine encoding both E6 and E7 can be used to provide protection and therapy against a tumour expressing HPV oncogenes. This study supports previously published studies that demonstrate an enhanced HPV-specific CTL response when both E6 and E7 are administered together in the context of a vaccine.
These results have generic implications for the design and administration of DNA vaccines encoding chimeric antigens. The results of this study have specific implications for the design of HBsAg-based DNA vaccines delivering HPV antigens for the protection and therapy of HPV-associated squamous carcinomas. An effective therapeutic vaccine designed to treat HPV associated cancers has the potential to dramatically reduce the burden of cervical carcinoma and associated death rate.

Pulmonary infection of mice with human metapneumovirus induces local cytotoxic T cell and immunoregulatory cytokine responses similar to those seen with human respiratory syncytial virus.
Karen A. Herd, Michelle Nelson, Suresh Mahalingam, and Robert W. Tindle

Human metapneumovirus (hMPV), is a major cause of upper and lower respiratory tract infection in infants, the elderly and immunocompromised individuals. Virus-directed cellular immunity elicited by hMPV infection is poorly understood, in contrast to the phylogenetically and clinically related pathogen human respiratory syncytial virus (hRSV). In a murine model of acute lower respiratory tract infection with hMPV, we demonstrate the accumulation of IFN-γ producing CD8+ T cells in the airways and lungs at day 7 post-infection, associated with cytotoxic T lymphocytes (CTL) directed to an epitope of the M2-1 protein. This CTL immunity was accompanied by increased pulmonary expression of Th-1 cytokines IFN-γ and IL-12, and anti-viral (IFN-γ) cytokines, as well as chemokines Mip-1alpha, Mip-1beta, Mig, IP-10, CX3CL1. There was also a moderate increase in Th2-type cytokines cytokines IL-4 and IL-10 compared to uninfected mice. At twenty-one days post-infection, a strong CTL response could be recalled from the spleen. A similar pattern of CTL induction to the homologous M2-1 CTL epitope of hRSV, and of cytokine/chemokine induction, was observed following infection with hRSV, highlighting similarities in the cellular immune response between the two related pathogens. . This project was conducted in collaboration with the Assoc Prof Suresh Mahalingam’s laboratory, University of Canberra.  

Enhancing Cancer Therapy and Immunity by Administration of HBV virus-like particles (VLPs) displaying TNF family ligands.
Scott Thomson, Robert Tindle

Tumour necrosis factor (TNF) family ligands (TNF-FLs) have been shown to have a range of powerful immunological effects in vivo, including immune enhancement, suppression and/or provision of essential cell survival signals for key immune responses elicited during cancer, infection and autoimmune diseases. In this project we will exploit hepatitis B virus (HBV) surface antigen (HBsAg)-based virus-like particles (HBVLPs) to deliver and display TNF-FLs, which could be used as:
a) Immunoregulatory agents in immunotherapies for cancer, autoimmunity and infection.
b) A direct anti-cancer therapeutic (in the case of HBVLPs displaying TNlF-alpha)
c) To enhance vaccine-elicited adaptive immune responses, e.g. the current HBV vaccine.
We will investigate the capacity of HBVLPs modified to display the extracellular region of TNF-FLs to augment T cell immunity and tumour therapy. The current HBV vaccine, based on HBVLPs (Engerix, GSK; Recombinvax HB, Merck), has been administered pan-globally to millions of recipients and has an excellent safety profile. To display TNF-FLs on the surface of HBVLPs we have developed a novel technology which evolved from our extensive research and experience using HBVLPs as a delivery platform for foreign antigen sequences. Our technology is based on two recent key findings in our laboratory: 1) That covalently linked HBsAg dimers can readily form VLPs and 2) That attaching the extracellular portion of TNF-FLs onto the C-terminus of HBsAg dimers is vastly superior (3x-15x VLP yield) compared to the same addition onto HBsAg monomers. These two findings mean it is now feasible to manufacture and explore the potential ofHBVLPs displaying TNF-FLs in vivo.
Initially the project will compare HBVLPs displaying; TNF-alpha, CD40L (CD154), 4IBBL, CD70, CD30L, and GITRL. Each HBVLP-TNF-FL will be tested to determine its impact on T cell immunity, when administered either alone or in adjuvant. This will enable us to select the HBVLP-TNF-FL which is the most potent enhancer of CD8+ cytotoxic T cell (CTL) immunity and use it to further characterise its immunotherapeutic potential to enhance the control of tumours in three different mouse tumour therapy models (melanoma, squamous cell carcinoma and TRAMP adenocarcinoma prostate cancer models). Additionally, we will examine the direct anti-cancer effects of HBVLP-TNF-alpha when delivered by intra-tumoural injection, in the same three mouse cancer models.
An important goal of this project is to overcome the current lack of an efficient and practical way to exploit TNF-FL signalling which is a critical barrier for the exploitation of these pathways in cancer applications. Being small molecules, the half-life of soluble versions of TNF-FLs in vivo is often short. To circumvent this problem relatively high local concentrations are generally required in order to produce useful effects. The high systemic doses used in the past have led to significant detrimental side effects, for example the side effects produced by TNF-alpha therapy, in early human cancer therapy trials. Our strategy will also avoid the well-known practical clinical limitations of using TNF receptor-specific agonistic antibodies or genetic-based ligand delivery strategies. A specific objective of the project will be to determine the immunological impact of delivering TNF-FLs concentrated on the surface of HBVLPs, which may enable lower, safer and more durable doses to be used in vivo.
The outcomes of the project will be: 1) Characterisation of the immunological impact of concentrating TNF-FLs on the surface of HBVLP-based therapeutics and 2) Insights into how they then might be used as potent immunoregulatory agents in immune-based therapies for cancer, as a direct anti-cancer therapy and potentially how they could enhance vaccines against infectious diseases.

Effects of Co-stimulatory Molecule CD70 on the Immunogenicity of Recombinant Hepatitis B Surface Antigen Vaccine.
Jason Cheong, Scott Thomson, Robert Tindle

The current vaccine for Hepatitis B Virus (HBV) infection consists of virus-like particles (VLPs), comprised solely of Hepatitis B surface antigen (HBsAg), which are non-infectious and highly immunogenic. Foreign DNA sequences have been incorporated into HBsAg to obtain chimeric vaccines with protective functions. Polyepitope extension sequences, encoding disease-specific cytotoxic T-lymphocyte (CTL) epitopes added at the termini of HBsAg-based constructs, have shown to induce strong CTL responses in immunised mice, correlating with disease protection. Recombinant DNA inserts of up to 395 amino acids have also been cloned onto the HBsAg C’-termini, and such constructs retain the capacity to form VLPs. Therefore, chimeric HBsAg-based vaccines have an enormous potential as an adaptable platform for delivering protective CTL epitopes.
The CD27/CD70 receptor/ligand pair has been shown to be influential for cell survival after initial activation of CD8+ cells, and appears to increase T-cell cytotoxicity. Hence, In this present study, we engineered soluble CD70 (soICD70) costimulatory molecule to be attached to the C’-terminus of an existing HBsAg-based chimera plasmid vaccine that encodes multiple disease-related epitopes. In a separate construct, the soICD70 was also tagged at the N’-terminus with an endoplasmic reticulum (ER) signal sequence. It was inserted downstream of a second CMV promoter, allowing its independent expression from the HBsAg molecule. Mice were immunised with both recombinant HBsAg constructs plus controls. Epitope-directed effector and memory CTL responses were quantified and compared against immune responses elicited from similar recombinant HBsAg vaccines without solCD70 co-stimulatory molecule expression. Tumour protection studies were also done to determine the extent of protection coverage, conferred by a soICD70-expressing vaccine. We hypothesised that CD70 expression would enable augmentation of the immune response, and therefore acquiring enhanced CTL activity and tumour protection.

We showed that both new recombinant HBsAg DNA vaccines encoding solCD70 expression did not induce augmented epitope-specific effector nor memory immune responses, compared to DNA vaccines without so1CD70. The tumour study also showed no enhancement of tumour protection. Further experiments showed the absence of solCD70 molecules within cells transfected with soICD70expressing plasmids. Thus, it is likely that protein stability or the inability of solCD70 to fold in such a configuration is a problem that needs to be addressed in the future to unlock the utility of co-stimulation ligands.