Hunting for the papilloma virus
S. M. Andreev,
Candidate of Chemical Sciences, SSC Institute of Immunology, FMBA of Russia
"Chemistry and life" No. 12-2008 (published on the website "Elements")
Just as hunters prepare for hunting – they study tracks, install feeders, place beaters, put shooters on the firing line, release dogs – so do immunologists, in order to rid the body of the virus, gradually prepare and direct the process of activating the immune system. The first action is to identify the target.
The target is a virus proteinThe papilloma virus, once in the skin cells, manifests itself as a two-faced Janus.
Its genome can be in two forms: either embedded in the chromosome of the cell, or exist as a free molecule – DNA episome. If it is embedded in the cell chromosome, then in the presence of predisposing factors, the program of its genome is implemented and the virus begins to multiply. The affected area turns into a papilloma, or wart. And sometimes it happens not on the arm or leg, but on the mucous membrane in the most intimate places of the human body. And if the virus belongs to a special subtype, then in ten years the affected cells may transform into a cancerous tumor.
For its successful life, the virus must force the cell to synthesize several proteins that are useful to it. In particular, the papilloma virus needs protein L1, which goes to build the shell of new viral particles, as well as proteins E6 and E7 – they make the infected cell immortal, these are oncogenic proteins.
There are several fundamentally different ways to fight the virus. The first is to prohibit it from binding to target cells, the second is to prevent it from multiplying in them, the third is to destroy diseased cells along with the virus. Since in the case of the papilloma virus, diseased cells do not contain it in the traditional sense – after all, after getting into the cell, only DNA remains from it, which sets the synthesis of those most dangerous proteins, it is impossible to prohibit reproduction. Therefore, the first and third methods remain in the hands of doctors, that is, a preventive vaccine and a therapeutic one. Recently, another one has appeared – the use of short, so-called silent RNAs that silence viral genes. But the movement along this path is still at the very beginning.
The situation is complicated by the fact that each mammalian species has its own papilloma viruses that do not live in the tissues of other species, and the human virus reproduces extremely reluctantly in cell culture. As a result, the search for a vaccine has to be conducted on mice, and then hope that these results can be reproduced in tests with the participation of volunteers.
A great success in the fight against the papilloma virus was the discovery in 1991 of an interesting phenomenon made by Zhou Jiang and Ian Fraser, who worked at the Princess Alexandra Hospital in Brisbane, Australia (Ian Fraser was awarded the Italian-Swiss prize of the Balzan Foundation in the amount of 1 million Swiss francs in November 2008 for this work. – Ed. note). They found that L1 proteins spontaneously assemble into virus-like particles (HPV) even if there is no genetic material inside them. Such proteins can be synthesized very simply and in large quantities with the help of yeast by embedding the necessary gene in them. They come out of yeast already in the form of ready-made particles, and their immunogenic properties are the same as those of a real virus. If HPV is injected into the body, then protective antibodies will begin to be produced in it. In many experiments, such protection has proven its reliability, and eleven years after the discovery, a large experiment involving 1,533 volunteers showed that such a vaccine against human papillomavirus subtype 16 (HPV16), firstly, is quite safe, and secondly, provides complete protection of the body. This result is also important because subtypes 16 together with 18, 31 and 45 account for 80% of all cases of tumor degeneration of virus-affected tissue, primarily cervical cancer. Another 13 varieties of this virus are responsible for the remaining 20%. In the six years that have passed since the beginning of the experiment with the participation of volunteers, the effect of the vaccine has not weakened – not a single case of infection with the virus among them has been recorded.
In 2006, Merck was the first company to receive permission to sell Gardasil preventive vaccine in the USA and Europe, which protects against papillomavirus infection of subtypes 6, 11, 16, and 18. Doctors recommend vaccination for girls aged 9 to 26 years. In 2007, GlaxoSmithKline received permission for the Cervarix vaccine, which protects against 16 and 18 subtypes of the papilloma virus.
Peptide BulletsHowever, these are all preventive vaccines, and it is not yet known how quickly the immune system will forget about the information it received at the time of vaccination.
Therefore, a therapeutic vaccine that destroys already infected cells is of great interest.
To create it, immunologists hope to use such a bright trace of the virus as those two proteins, E6 and E7. They must necessarily be on the surface of diseased cells, and, therefore, the task is to teach killer cells to find these traces.
Many laboratories in the world compete in this field, because the price of victory is very high. To excite the immune response, E-proteins themselves, and their peptide fragments (T-epitopes), and chimeric constructions of E- and L-proteins are used. (Recall that substances that excite the immune response are called antigens.) To enhance the response, cytokines, heat shock proteins and other stimulating elements are added to the vaccine.
An indispensable component of the vaccine is a vector that delivers antigens – E–proteins or their peptide fragments - to special cells of the immune system (macrophages, dendritic cells), after which they begin to produce special cytokine proteins and give instructions to killer cells that they, in fact, need to destroy. Vectors for papillomavirus antigens in different experiments were smallpox vaccine virus, adenovirus, alphavirus, bacteria. It should be noted that dendritic cells are the most active partners for stimulating a strong immune response, so it is desirable that antigens get into them.
That is why much attention is paid to chimeric constructions based on heat shock proteins, that is, containing both E-protein and an immune response booster protein. The heat shock protein has an affinity for dendritic cells. It was by this method in model experiments that it was possible to record a decrease in the size of the tumor with a single immunization. Currently, several candidates for therapeutic vaccines are at various testing stages, but unlike preventive vaccines, so far none of them has yielded acceptable efficacy. The idea arises that success can be achieved by improving all parts of the vaccine: peptide fragments that most correctly repeat the T-epitopes of the E-protein, a vector for targeted delivery of the vaccine to the dendritic cell, an immune response stimulator, as well as the vaccine carrier on which all this is supported.
The work on the implementation of the program "Combined vaccine to HPV 16, 18 and 31", which was led by the teams of two institutes – the Institute of Immunology and GOSNIIGenetics - led by Academician M. R. Khaitov, we started with computer calculations. To conduct such a study, it is necessary to have good tools – a set of specific antibodies to various types of virus, reference drugs (viral proteins), so that there is something to compare the products obtained. In Russia, none of this could be bought, and even in the USA it was impossible to buy antibodies to some L1 proteins - no company made them for sale. And here Professor Neil Christensen from Milton Hershey Medical Center in Pennsylvania helped us a lot by providing micro-samples of some recombinant proteins and monoclones. But we had to synthesize a lot ourselves, including antibodies to three types of L1 and E7 proteins and to their various sites, which were calculated using computer algorithms. All peptides were synthesized chemically by the solid-phase method, there were about 20 of them. Such peptides are safe for the body, and it is not difficult to synthesize them, but the immune system does not react actively enough to them – pure peptides almost do not induce antibodies. Therefore, it is necessary to combine them, firstly, with the carrier, and secondly, to add substances that stimulate the immune system. Hemocyanin (a huge protein from a snail), "polyoxidonium", on the basis of which the "Grippol" vaccine was previously created, and some other stimulants were used as carriers.
In parallel, three types of L1 proteins were synthesized in yeast in the State Genetics Institute (there was a problem with one of them), which was assembled into the correct virus-like particles and reacted correctly with antipeptide antibodies and reference monoclonal antibodies.
The responses to different peptides were very different, and these experiments revealed the most effective fragments of both L1 and E7 protein. The final test showed that if snail hemocyanin or a special immunostimulator PM is used as an adjuvant carrier to these peptides, then the immune system reaction will be the strongest. The most important thing is that this produces a strong immune response to L1, and also activates populations of specific killer cells, the so-called cytotoxic CD8+ T cells. It is they who must destroy infected cells. In fact, the data obtained give reason to believe that we have managed to create separate components of a prototype of a combined vaccine, preventive and therapeutic. While this is not yet a vaccine, the drug should be tested on an acceptable biological model, for example, on mice with a transferable tumor caused by oncogenic protein E7. Moreover, the resulting vaccine preparations are more focused on prevention, since it has already been proven that the L1 protein generates a strong protective response even in the absence of an adjuvant.
Peptides are weak immunogens, we were convinced of this by making a conjugate of one peptide from the E7 protein with a standard Freund adjuvant. Activating T cells in this way is not easy, and probably you need to change tactics. First of all, this requires targeted delivery of peptide antigens from the E-protein to dendritic cells in order to achieve specific and strong activation of killer CD8+ lymphocytes.
And then we decided to apply a new approach: to use fullerenes, whose high cell-penetrating ability is well known, as a delivery booster, and to add a vector that would direct them to dendritic cells. To do this, I had to do work that was not directly related to the creation of the vaccine: to determine the immunogenicity of fullerene and their derivatives with amino acids and peptides, as well as their ability to penetrate into cells. And here we got very interesting results.
Fullerene and lifeA lot has already been said about the toxicity of fullerenes, and there are two opposing points of view: "they are extremely harmful" and "they are very useful."
It is possible that the appearance of such different points of view is facilitated by the peculiarities of the physicochemical properties of fullerenes, namely the fact that they are not soluble in water due to their hydrophobicity. As a result, in order to prepare a drug and inject it, for example, into the blood of an experimental animal, the fullerene molecule needs to be modified somehow: attach hydrophilic groups to it or add surfactants to the solution that can suppress the hydrophobicity of fullerenes. These components may have specific chemical activity, they turn out to be toxic in themselves, which is quite capable of leading to a conclusion about the toxicity of fullerene itself. And such cases are widely known. For example, one of the very authoritative experts on the study of the biological action of fullerenes, G. V. Andrievsky from the Institute of Therapy of the Academy of Medical Sciences of Ukraine, proved that the data given in the most frequently cited article on the toxicity of fullerenes are associated with an artifact: a toxic substance, tetrahydrofuran, was present in the studied fullerene–containing liquid. G. V. Andrievsky himself is known for having managed to create a technique for obtaining a fairly concentrated solution (nanosuspension) of fullerene in water.
The problem of toxicity of fullerenes and other nanoparticles has long been very relevant. Immediately after the discovery of fullerenes, the idea spread among scientists that these molecules could be useful as a means for transporting medicines. Now hundreds of fullerene compounds have been synthesized, many of them exhibit biological activity, but it has not come to the creation of commercial drugs. Perhaps because they have no special advantages over non–fullerene analogues, or perhaps because of a lack of deep understanding of the mechanisms of metabolism of fullerenes and their interaction with living cells.
However, due to the development of nanotechnology, it is almost inevitable that conditions arise for environmental pollution with these very persistent compounds, the scale of production of which is increasing. It's time to solve the issue of the danger or safety of fullerenes, including immunological safety. First of all, we are talking about the ability of fullerenes to cause an immune response, for example, allergies.
Indifferent fullereneIn our experiments, we used a crystalline fullerene called fullerite, a nanosuspension of hydrated Andrievsky fullerene, as well as fullerene compounds with various amino acids, peptides and proteins.
In this case, the amino acids were attached directly to the fullerene ball. A method for obtaining such derivatives was developed back in 1994 at the INEOS RAS named after A. N. Nesmeyanov. There, in collaboration with the Institute of Problems of Chemical Physics of the Russian Academy of Sciences, another functional fullerene was created, which is very quickly sewn to peptides and proteins containing the amino acid cysteine. Why was it necessary to obtain such complex compounds?
The fact is that 12 years ago we already tried to induce in mice a specific immune response to pure fullerenes and their amino acid derivatives and did not succeed at all in this matter. However, in 1998, there was information that one American group managed to achieve an immune response to fullerene in the presence of a strong immunostimulator. In our experiments, we just wanted to test this result, and as immunostimulants we took well-known allergens like egg and serum albumin. However, the result was the same: no specific response to fullerene proper was observed. But we found a well-expressed reaction to amino acids sewn to fullerene. (The response to pure amino acids does not develop at all, the body is tolerant to them.)
The lack of an immune response to fullerene can be explained as follows. Theoretically, in an aqueous medium, hydrophobic fullerene molecules cannot exist in a single state, but gather in clusters of tens or even hundreds of molecules. Once in a living organism, these clusters must interact with hydrophobic components of the medium and electron–donating molecules - proteins, fats or amines. As a result, the carbon sphere can be completely closed by these molecules, and then it is not able to bind to the B-lymphocyte receptor, which gives a signal for the development of an immune response. However, in itself, the presence of a specific fullerene receptor in B cells raises serious doubts. Fullerenes as artificial molecules were obtained quite recently, in 1991, and in the process of evolution organisms could not come into contact with it, therefore, it is unlikely that there are cell clones that recognize such molecules. As the work on the creation of carbon endoprostheses showed back in 1979, it is impossible to obtain antibodies to other forms of carbon – graphite and diamond. Although such an enzyme as HIV protease is known, whose active center has a hydrophobic cavity: fullerene (with hydrophilic suspension) fills it well and thereby blocks the activity of the virus. But the receptors on the B-lymphocyte for catching foreigners are located outside, that is, they are turned into an aqueous medium, have a hydrophilic nature and are unlikely to capture fullerene.
Like a knife in butterIf fullerene does not cause an immune response, can it somehow damage the cell?
The answer to this question is given by a series of experiments conducted by us with erythrocytes, human platelets and symbiosomes – products of the symbiosis of legumes with nitrogen-fixing bacteria of the genus Rhizobium. The fact that fullerene has penetrated into the symbiosome can be judged by the charge of its membrane. In the presence of ATP and magnesium ions, it is able to generate a positive charge on the inside of its membrane. Fullerenes with amino acids sewn with proline or aminocaponic acid are negatively charged. Once inside the symbiosome, they neutralize the charge on the membrane, which can be detected by spectral methods using special probes. As it turned out, this process is very fast: when a solution with fullerene derivatives was added, the cell membrane instantly lost its accumulated potential.
Fullerene with another acid, arginine, on the contrary, acquires a positive charge, and therefore its effect on the symbiosome could not be noticed. But it manifested itself on red blood cells, the membrane of which was negatively charged with valinomycin (K+ leaves the cell at the same time): with the addition of C60-Arg, a rapid discharge of potential occurred.
The change in the membrane potential was not the only effect. There is such a fluorescent dye – acridine orange. It changes its glow when the acidity of the medium changes. With its help, it was possible to additionally confirm that amino acid derivatives of fullerenes actually easily penetrate into cells and change the acidity of the medium.
We also confirmed that fullerenes easily penetrate into different types of cells. For example, there is a lot of calcium in platelets, so it is possible to study the transport of fullerene with the help of another dye, chlortetracycline, the glow of which depends on the concentration of calcium ions: if fullerene interacts with it, it extinguishes this glow. And so it turned out: when fullerenes, including Andrievsky fullerene, were added to platelets loaded with chlortetracycline, fluorescence quenching was observed. However, it turned out that Andrievsky's fullerene enters the cell a hundred times slower than with amino acid derivatives.
So, it has been established that fullerene, due to its hydrophobicity, passes freely enough through the lipid membrane of the cell. This gives rise to an idea that many have already heard: a fullerene with a fixed peptide can drag it inside the cell. This means that it can serve as an excellent means of delivering peptides to the dendritic cells of the immune system.
To test this assumption, we first attached to fullerene the peptides of the E7 protein found at the previous stage, which cause the greatest immune response. Secondly, a vaccine carrier was synthesized based on a copolymer of vinylpyrrolidone and maleic anhydride, to which chains of fatty acids were attached. Fullerene molecules with peptides clung to these hydrophobic tails due to van der Waals bonds. The result turned out to be very good already in the first experiment. The drug really behaved as a therapeutic vaccine should, generating specific T cells and antibodies. But so far we have not been able to trace in detail the mechanism of its action. Unfortunately, due to the termination of funding, this work had to be interrupted. Although we do not lose hope for a continuation, but the time is lost, and foreign researchers are not standing still.
Work started on the initiative of
Academician of the Russian Academy of Sciences R. V. Petrov
Portal "Eternal youth" www.vechnayamolodost.ru08.04.2009