Field of Science

Can we use viruses to vaccinate against - and cure - established cancers?

Everyone is aware of the ability of our immune system to defend against microbial pathogens yet its role in the prevention of other diseases - like cancer - is generally over-looked. Yet it is through the harnessing of our immune system that novel ways of combating cancer may arise. And interestingly enough, through the use of engineered viruses - the same ones our immune system protects against - we may now control aspects of immunity to suit these medical needs. Kotke et al, report in Nature Medicine just this week, their use of a new virus-based immunotherapy platform that was able to effectively 'cure' mice suffering with cancer.

One of the hallmarks of cancer appears to be the ability to persist in the face of an active immune system - see fig. 1. Newly cancerous cells and tumours are able to survive and proliferate without - or at least protecting themselves - against a full-blown immune attack. Our immune system is usually able to protect us from the development of cancer but in some cases something fails and the result is more often than not - cancer.

Fig 1. Newly recognised cancer hallmarks - note avoiding immune destruction. (Hanahan and Weinberg 2011).

Through the recognition of 'tumour antigens' (proteins expressed only on cancer cells) or 'tumour-associated antigens' (proteins expressed differently on cancer cells), our immune system is usually able to mount an effective response toward those cells. This is the  system that tumours are able to suppress yet we may be able to boost the natural immunological nature of tumours in order to cure them. The discovery of these proteins - just like those found on the surface of virus particles or bacterial cells - may allow us to effectively vaccinate people against cancer, allowing their own immune system to remove the cancerous cells.

This is what we try and achieve through cancer vaccines and immunotherapies. Kotke et al set about trying to improve upon these current immunotherapy platforms, which - as they state - suffered from a number of problems including: lack of known tumour antigens and coverage of only a few such proteins. Most current immunotherapies rely upon the immunization with only a single antigen. Previous work by the group showed that if you kill normal cells from a patient in vivo - you may be able to elicit an effective anti-tumour immune response through the induction of tumour-associated antigen immunity (when cells die they either burst and release their insides).This work effectively showed that you could immunize with a wide range of tumour-associated antigens from normal cells and protect against cancer - both circumventing the above two problems.

VSV particles -
To improve upon this model, they developed a virus-based platform for the expression of a wide range of tumour-associated antigens in vivo termed altered self antigen and epitope library (ASEL) - see fig 3. They based their method upon the vesicular stromatitis virus - or VSV - normally a virus solely of livestock that also has the ability to infect humans. In humans it causes a generally mild flu-like illness and may form vesicles on the skin. Although a single-stranded non-segmented negative sense RNA virus, we have the ability to generate infectious VSV particles entirely from cDNA plasmids encoding the entire VSV genome. This has facilitated the development of VSV as a key eukaryotic expression vector for multiple uses, such as these cancer immunotherapies and specifically, as an oncolytic treatment. Using standard molecular biology techniques (PCR, restriction enzyme digests and ligations) you can insert any gene from whatever source you want into the VSV genome and it will be expressed inside cells following infection. The benefit with using this virus is that even without the expression of tumour antigens from it's genome, replication within a cell will kill the cell anyway. It is a double hit strategy.

Fig.3. Cloning the cDNA library into the VSV genome in forward and reverse orientations = VSV-ASEL library

In order to express hundreds of tumour-associated antigen genes, the group used reverse-transcriptase PCR to amplify all expressed genes from normal prostate tissue and inserted the entire normal prostate tissue cDNA library into VSV. They were then able to infect mice that suffered with prostate cancer and observe what happened to their tumours - specifcally, was an effective immune response generated and did the tumour shrink? VSV virus particles were injected into the mice, virus entered the cells of the mice and began to replicate and express their genes, including the newly inserted prostate cDNA. Essentially, thousands of virus particles were adminsired, each containing a slightly different gene from the prostate cDNA library. High levels of tumour associated antigens were therfore being expressed in mice allowing for the generation of an effective immune response.

Survival of mice treated with the VSV viruses - GFP expressing negative control; and the VSV ASEL in mice with established 'TC2' prostate tumours.

This approached effectively cured the mice who suffered from prostate cancer. Following this treatment a number of resistant tumours emerged which were again subjected to a further treatment using a cDNA library taken from the tumour itself this time and this readily treated the secondary resistant cancers. The clinical benefits of this approach can hardly go unnoticed. The ability to administer a broad tailored therapy that has the potential to cure an established tumour will be revolutionary, especially given the relative ease at which this can be developed 'at the bedside'. The ability to easily genetically manipulate viruses has - and will continue to - revolutionise the medical sciences. Look out for the eminent clinical trials. 

ResearchBlogging.orgKottke, T., Errington, F., Pulido, J., Galivo, F., Thompson, J., Wongthida, P., Diaz, R., Chong, H., Ilett, E., Chester, J., Pandha, H., Harrington, K., Selby, P., Melcher, A., & Vile, R. (2011). Broad antigenic coverage induced by vaccination with virus-based cDNA libraries cures established tumors Nature Medicine DOI: 10.1038/nm.2390

Improving global immunisation with more heat tolerant vaccines

During the last century, mass vaccination campaigns were rolled out across much of the industrialised world. And, with much success, we have near eradicated most common virus infections from these populations.   Despite the more recent efforts to bring the developing world into this fold, major setbacks have been uncovered, one being: how exactly are we to stably transport  delicate vaccine stocks from their place of production to the regions that need it most.

   Many of these vaccines, known as 'live attenuated vaccines' - or LAVs-are generally unstable in the environment outside of our cells and bodies. For example, the measles virus has an outer lipid membrane that is highly delicate and damage to this removes any chance of this vaccine working. Basically, these LAVs, which rely upon a weaker infection, cannot successfully complete their replication cycle without an envelope. Generally, the ability of a vaccine to provide protection is intrinsically linked to its overall structure; you destroy the structure, you destroy protection.

Unstable measles virus particles
  The more difficult places to transport vaccines to  happen to be more tropical regions, like sub-Saharan Africa, South America and South-East Asia. These places are very warm and humid which goes against what our vaccines like. With temperatures up to 40 degrees Celsius and the fact that freezing also destroys the vaccine, the only way forward is the implementation of a cold chain. If we keep the vaccine stock 'chilled' from manufacture (see Merck or the Serum Institute of India) all the way to administration, then we have a chance to prevent its degradation. But this is not as easy as it may sound as it is economically and logistically difficult to achieve this in developing countries. To overcome this, the produced vaccine stock is freeze-dried to remove any water that may contribute to its instability and when this prep reaches the clinic it is then reconstituted through the addition of a solvent preparation, which has then all got to be kept cool.

With measles virus vaccines however, even upon reconstitution, there are still major losses to immunogenicity and this problem is compounded with the use of multi-dose vials of vaccines. A single vaccine stock, prepared in the morning may now sit in the clinic unrefrigerated until the end of clinic hours. If we were then to get immunized at 5:00 pm, what are the chances of that being successful? It is thus not much of a surprise to hear that many virus outbreaks have been attributed to breaks in the cold chain like this. Our ability to eradicate many diseases in these countries and hence worldwide, is blocked by difficulties in the cold chain.

   Being so, Bill and Melinda Gates identified "Vaccines That Do Not Require Refrigeration" as one of their funded 14 Grand Challenges in Global Health and the results of one potential solution to this problem have just been published in the journal Vaccine (read it here). This work aimed at identifying novel reconstitution liquids that would increase the thermostability of the prepared vaccine and therefore increase its 'shelf-life' in these more difficult environments. Using a recombinant measles vaccine virus that expresses green fluorescent protein (GFP) upon infection, the group were able to assess the level of infectivity of a range of vaccine stocks reconstituted in varied solutions.

Counting measles virus infected (GFP positive) cells

   Indeed, they tested >11,000 formulations (myriad combinations of buffers, stabilizers, solubilizers, preservatives, pH and tonicifiers - whatever they are?) using their recently developed high-throughput system in which they were able to automate the whole process. These results were validated and confirmed with another virus, this time an adenovirus expressing GFP; results were the same. The group identified a formulation that caused the vaccine to "suffer <1.0 log loss after 8 h at 40 ◦ C in the liquid state", a major improvement on a previous loss of" 1 log of potency after 8 h at 37 ◦ C in the reconstituted (liquid) form". In conclusion, they identified a novel vaccine formulation that substantially increased the stability of two viruses, which can be used in immunization campaigns worldwide. As I've alluded to before, the use of high-throughput screens has much potential in the world of virus research.
Log loss of infectivity of vaccines

   How is this result likely to change vaccine implementation in the real world then? Well, the evidence presented here indicates the ability of creating more stable vaccines that would transfer to better vaccine coverage yet the real problem appears to be whether current vaccine manufacturers would accommodate such a significant change to production, especially given their inherent unwillingness to invest in the developing world.

   The cost associated with this may be offset by the savings made through reduction in vaccine wastage and loss of cold chain implementation but we must realise that only when such an improvement looks attractive to the market will change come. The future of vaccination looks to be bright, especially with the combination of newer technologies and the backing of large philanthropic organisations.

ResearchBlogging.orgSchlehuber LD, McFadyen IJ, Shu Y, Carignan J, Duprex WP, Forsyth WR, Ho JH, Kitsos CM, Lee GY, Levinson DA, Lucier SC, Moore CB, Nguyen NT, Ramos J, Weinstock BA, Zhang J, Monagle JA, Gardner CR, & Alvarez JC (2011). Towards ambient temperature-stable vaccines: The identification of thermally stabilizing liquid formulations for measles virus using an innovative high-throughput infectivity assay. Vaccine PMID: 21616113