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Feature

A Multipurpose Vaccine

The world's most common vaccine could be even better.

Glenn Buchan and Margaret Baird

When the body is invaded by foreign organisms like viruses, bacteria and other microbes, it sets up a defensive reaction to eliminate them. This immune response involves cells such as lymphocytes, phagocytes and specialist antigen presenting cells (APCs).

This first response to infection takes time to develop. Prokaryotic, or single-celled, organisms (such as bacteria) multiply much faster than eukaryotic, multicellular organisms (such as us): the average doubling times are 20 minutes and 24 hours respectively per cell. Thus highly virulent organisms can overwhelm the developing immune response, causing disease.

An interesting aspect of the immune system is that it has memory. Pre-exposure to an antigen primes the system so that it can respond more rapidly and more strongly the next time it is challenged by the same antigen.

This characteristic is exploited in the process of vaccination, where the body is purposely exposed to the antigen in order to establish immunity. The type of immune response developed is specific to the type of antigen, and is highly controlled by regulatory cells of the immune system which communicate by secreting various chemical signals.

However, incorrect signalling can lead to ineffective responses that are incapable of preventing disease. Another problem is that the length of memory can be variable. Whereas two doses of measles vaccine early in life can protect individuals for more than 70 years, other vaccines, such as tetanus or meningococcal, protect for much shorter periods (3-5 years) and require regular boosting.

Basic Immune Response

The induction of an immune response requires four components:

  • an antigen, a substance that provokes an immune response; typically a virus or bacteria, but sometimes other substances
  • antigen presenting cells, which break down large molecules or microbes into smaller peptide molecules (antigens) which are then displayed on the surface on the APC to stimulate the immune response
  • costimulatory signals (eg cytokines)
  • T cells with receptors that can recognise and respond to the antigen when they also receive a costimulatory signal

Two major groups of regulatory T cells have been defined. Type 1 (T1) responses result in the activation of cytotoxic (killer) T cells and activated macrophages (leukocytes, or white blood cells, which scavenge unwanted material).

T1 or cell-mediated responses are required for protection against intracellular microbes such as mycobacteria and some viruses which infect and hide within cells of the host. These cells need to be killed by the immune system to release the microbes so that they can be killed by the immune response. T1 cells are also important in the killing of tumour cells.

Type 2 (T2) responses result in the production of large amounts of antibody, which stick to microbes and target them for destruction by phagocytes. Antibodies are important for immunity to microbes or toxins which exist for a significant period outside the host cell.

The induction of a T1 or T2 response is regulated by a series of signals delivered by immune hormones called cytokines. Cytokines important in Type 1 responses include interferon gamma (IFNg) which activates macrophages, and interleukin 2 (IL-2) which activates and increases the numbers of cytotoxic T cells. Cytokines such as interleukin 4 (IL-4) are important in the induction of a Type 2 response, as they act on B cells to induce antibody production. The T1 cytokines seem to suppress the production of T2 cytokines and vice versa. Therefore, it is important that any vaccine induces the right type of immune response to protect against a particular microbe. The result of activation of an inappropriate type of response will be vaccine failure, leading to disease if infection occurs at a later date.

Several factors can have an impact on the ability of a vaccine to protect against disease.

Genetic and environmental factors can cause aberrant regulation, resulting in the wrong response or the development of an inadequate response. Live vaccines are known to induce good long-term memory, whereas killed vaccines or parts of microbes induce poor memory responses which give short-term protection from disease.

Thus, a number of factors are important to guarantee a vaccine will induce a long-lived protective immune response. These include:

  • the use of a live, attenuated (i.e. weakened) organism
  • the induction of the right cytokines, which in turn will induce the right type of immune response (T1 or T2)
  • the efficient delivery of the vaccine to the APC so that the T-cells are activated properly

We are investigating better ways to construct and deliver vaccines using the vaccine against tuberculosis, BCG. We hope to increase the effectiveness of vaccination by manipulating the signals within the immune system to maximise the specific response to particular antigens.

A Multipurpose Vaccine Figure A (7KB)

BCG Vaccine Vector

Bacillus Calmette-Guerin (BCG) is an attenuated form of Mycobacterium bovis, and is the most widely used vaccine in existence. It was developed to immunise humans against tuberculosis, and has been used successfully in many countries. The widespread use of BCG has confirmed its status as the safest vaccine we have. Over 3 billion doses have been dispensed since 1948, and it is estimated that one-third of humanity has been vaccinated with live BCG. The incidence of serious complications are 0.19 per million and it costs 6 cents per dose. All of these factors make it an excellent candidate as a general vaccine vehicle.

There are technical problems involved in the use of BCG. BCG, like all acid-fast bacteria, has a cell wall rich in lipids, or fats. It clumps easily and will not form single colonies without detergent present in the growth media. It is difficult to get DNA into BCG using standard methods as compared with E. coli. In addition, our knowledge of the molecular biology of BCG is relatively primitive.

Our approach has been to exploit the natural ability of BCG to act as a powerful stimulant of the immune response, and improve it by inserting genes which encode for antigens and/or cytokines. We are looking at ways to genetically engineer BCG so that it contains both a specific antigen and the necessary cytokine signals required to "turn on" the correct immune response.

Other laboratories have investigated the use of BCG to express antigens derived from a number of microorganisms, and these have been used to immunise experimental animals against disease.

In collaboration with Dr Mike O'Donnell at Harvard Medical School, we are taking this a step further to develop an integrated vaccine which will deliver antigens as well as cytokines.

To achieve this, our collaborators at Harvard have designed a shuttle plasmid capable of shuttling between E. coli and BCG. This plasmid contains two multiple cloning sites, and the genes cloned into the two sites are under the control of separate high-efficiency, heat-shock promoters that work in both types of bacteria.

Thus new constructs can be developed in the well-characterised, rapidly growing E. coli system before being transformed into the slow-growing BCG.

We are developing recombinants (designated as rBCG) that can express:

  • two separate cytokines, one of which upregulates T cell functions while the other upregulates APC activity
  • a cytokine and an antigen independently
  • antigen/cytokine fusion proteins

The latter are of particular interest, as the fusion of antigen and cytokine has been shown to be more effective than when each is given independently. Initially, we will use an rBCG engineered to express the well-characterised viral influenza haemagglutinin antigen (HA) fused to one of a variety of cytokines. This will allow us to investigate whether inoculations of rBCG expressing HA and a cytokine can influence the type of the immune response generated to the HA antigen, and whether this can be used to induce superior protection against influenza in mice.

Antigen Presenting Cells

As mentioned before, the induction of an immune response requires that the antigen is presented on specialised antigen presenting cells. APCs respond to infecting microorganisms and transmit signals which activate T cells. These signals can be regulated by cytokines in the extracellular environment, biasing the direction of the immune response. The two major classes of APC are dendritic cells, essential for the initiation of an immune response, and macrophages, which are important in amplification and in memory responses. Macrophages may also process antigen for dendritic cells to present. Both these APC produce cytokines that affect the type of immune response that occurs.

We are looking to explain how dendritic cells and macrophages function and interact after they have been infected with rBCG/HA/cytokine constructs. Directing antigen to particular APCs has been shown to alter the degree and the direction of the immune response.

Recently, inoculations of dendritic cells pulsed with antigen have been used as an immunotherapy against cancer. We propose to determine whether dendritic cells pulsed with rBCG/HA/cytokine constructs can further enhance the immune response to HA antigen. This will indicate whether it is feasible to use these to immunise against disease.

Superior Tuberculosis Vaccine

We have already begun to look at the ability of rBCG to induce superior immune responses against tuberculosis. Tuberculosis, the disease associated with Mycobacterium tuberculosis and Mycobacterium bovis infection, is responsible for over four million deaths annually. We have tested a prototype rBCG vaccine which secretes the immune regulatory cytokine interleukin 2 (IL-2). The rBCG/IL2 is designed to improve protective Type 1 responses, particularly in susceptible animals.

Two strains of mice, one susceptible and one resistant to tuberculosis, were vaccinated subcutaneously with either live BCG or live rBCG. The degree of lymphocyte activation was measured in response to antigen from Mycobacterium tuberculosis. Resistant mice produced moderate levels of lymphocyte activation to both vaccines. The T lymphocytes of susceptible mice were strongly activated by the rBCG and multiplied two to three-fold more than the lymphocytes from the resistant strain which had been exposed to rBCG.

This suggests that genetically susceptible individuals may gain more benefit from this new generation of vaccines than resistant individuals. These are the individuals that vaccine campaigns tend to miss. If these findings can be translated into humans then this could represent a significant advance in our ability to maximise vaccine coverage.

By 16 weeks, a classic Type 1 response typified by high levels of IFNg production and undetectable levels of IL-4 had developed in response to rBCG in both resistant and susceptible strains of mice. In contrast, the immune response elicited by the normal BCG waned dramatically by 16 weeks. In both strains of animals the IL-2 secreted by the rBCG increased the production of IFNg and maintained this level over a 16-week period.

This supports the hypothesis that the rBCG elicits a strong Type 1 response and also suggests that the rBCG is capable of improving the longevity of the immune response to the vaccine. The induction and maintenance of antigen specific Type 1 lymphocytes is important in sustaining a protective immune response to tuberculosis.

Our studies have also shown that the shuttle plasmid is stably maintained within BCG. Both in vitro and in vivo studies have shown that the rBCG continues to secrete the cytokine for up to three months in the absence of antibiotic selection. Evidence in the mouse model suggests that while the rBCG is more rapidly eliminated from the host, it still induces a superior immune response to the parental strain.

Improved Potency Enhanced Immunity

The most effective safeguard against infectious disease is vaccination. The ability to simultaneously improve the potency of vaccine antigens as well as direct the host's immune system towards the most effective response will be essential for the rational development of new vaccines.

The immune response is regulated by a complex network of positive and negative signals exerted by cytokines and APCs. Our challenge is to understand how to manipulate these signals to ensure that the immune system responds to each antigen in the most appropriate manner to optimise its protective capacity. Refining the conditions under which antigen is delivered to the host could result in more effective protection against a range of infectious organisms, tumour antigens and even allergies. The use of antigen fused to cytokine and expressed in BCG should result in more rational vaccine design.

Historically it has been thought that vaccines can only be effective in preventing disease, not in curing established disease. Our increasing understanding of the immune response and how it is regulated means that this concept is being increasingly challenged. There is growing evidence that, if formulated properly, vaccines can activate the immune response to eliminate or reduce pre-existing infection. Such vaccines will be able to eradicate disease much more quickly and effectively. The information gathered from these studies will allow us to understand the critical elements required of such a vaccine.

The purpose of our studies are therefore to not only improve existing vaccines but to challenge existing theories that have limited the development of new vaccines against pathogens that continue to threaten so much of the world population. BCG has been a staunch friend of humanity for over 70 years. We hope that its offspring will continue to act on our behalf well into the next millennium.

Margaret Baird works in the Department of Microbiology at Otago University.
Glenn Buchan works in the Department of Microbiology at Otago University.