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How Far Away Are We From A Heart Attack Vaccine?
| The First Immunization Studies
There was of course also the possibility that that oxidized LDL was attacked not only by the innate immune responses but also became a target for the adaptive response. Indeed, in 1989 Wulf Palinski, Joe Witztum and their coworkers at University of California San Diego identified presence of antibodies against oxidized LDL in human serum demonstrating for the first time presence of an adaptive, autoimmune response against oxidized LDL in man.
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| The presence of oxidized LDL specific T cells was subsequently demonstrated both in the circulation and within atherosclerotic plaques. Evidence was now mounting that atherosclerosis was an autoimmune disease or at least had an important autoimmune component. Was this autoimmunity the cause of atherosclerosis? This question was addressed by the San Diego group as well as by a joint research team with Jan Nilsson in Sweden and Prediman K. Shah at Cedar-Sinai Medical Center in Los Angeles by immunizing hypercholesterolemic rabbits with oxidized LDL. If atherosclerosis indeed was caused by an autoimmune responses against oxidized LDL this treatment would likely result in a much more rapid disease progression. The researchers were stunned by the results of their studies. Rather than getting more disease the immunized animals appeared to develop a protection against atherosclerosis and progression of the disease was significantly reduced. The results were subsequently confirmed by a number of groups using different animal models of atherosclerosis. It was quite clear that the autoimmune response against oxidized LDL represented a previously unknown endogenous defense against the development of atherosclerosis. |
| Development of Pilot Atherosclerosis Vaccines
A first challenge in the development of an atherosclerosis vaccine was finding the right antigen on which the vaccine should be based. One possibility would be to use complete oxidized LDL as in the initial animal experiments. However, this was considered a risky approach for several reasons. Oxidized LDL is a complex structure likely to contain a mixture of large number of different and poorly defined antigens. It could not be excluded that immune responses against some of these antigens could activate disease instead of protecting against it. Because of this it became important to identify and characterize the structures in oxidized LDL that were targeted by the immune system.
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| The scientist approached this problem in several ways, but a common theme was the assumption was that these immune responses were part of body's natural protection against atherosclerosis or alternatively were actively involved in the disease process. In San Diego Joe Witztum, Wulf Palinsky and their coworkers isolated antibody-producing B lymphocytes (so called B cells) from the spleens of atherosclerotic mice. These B cells were transformed to immortal cell lines (hybridomas) producing large amounts of antibodies. A large number of these hybridomas were found to produce antibodies that recognized structures in present oxidized LDL. One unexpected finding was that all of the antibodies belonged to the IgM family. IgM are the first antibodies produced in response to an infection and their production can be activated within hours (?). However, their specificity is poor and they can be said to represent a part of the immune system that is somewhere in between the blunt innate and the highly specific adaptive immune response. Most of the antibodies were found to recognize oxidized phospholipids present on the surface of oxidized LDL (figure 4). When the gene encoding these antibodies was sequenced another unexpected observation was made. The antibodies were identical to a commonly expressed antibody called T15 known to be an important part of the immune defense against bacteria called Streptococcus pneumoniae. This observation opened interesting possibilities because a vaccine against Streptococcus pneumoniae was already used clinically. When the scientist used this vaccine to immunize atherosclerosis-susceptible mice they developed high levels of IgM against oxidized phospholipids and the development of atherosclerosis was reduced by about 30%. Accordingly, immunization against Streptococcus pneumoniae or other antigens that increase IgM against oxidized LDL phospholipids represents one possible approach for development of an atherosclerosis vaccine. |
| Jan Nilsson, Prediman K Shah and their co-workers used another strategy to identify the antigenic structures in oxidized LDL. Their approach was based on the act that most immune responses are directed against proteins and more specifically against 8-12 long amino acid sequences in proteins (proteins are built by chains of amino acids linked together in sequences that are specific for each protein). The LDL particle contains only one protein, the 4500 amino acid long apolipoprotein B-100 (apo B-100) that encircles the LDL particle (figure 4) and that contains the binding site for the LDL receptor expressed by cells in need of increasing their cholesterol supplies. When LDL is oxidized this large protein is broken down into smaller fragments (or peptides) that get contaminated by the attachment of small and extremely reactive molecules called aldehydes. These aldehyde-modified peptide fragments of apo B-100 represent another possible target for immune responses against oxidized LDL. Hunting for immune responses against these structures the scientists constructed a library of aldehyde-modified peptide fragments that together matched the complete amino acid sequence of apo B-100. They then studied if they could find antibodies in human plasma that recognized any of these peptide fragments. After analyzing plasma from a large number of individuals they could identify antibodies against about one hundred different apo B-100 peptide fragments (figure 4). Many of these antibodies were of the less specific IgM type but interestingly several were also of another type called IgG. IgG antibodies are much more specific than IgM and their production requires involvement of specific T cells and re-arrangements of antibody coding genes in the B cells. |
| Could immune responses against any of these peptide fragments be the ones that were responsible for the protective effects observed in the previous animal studies? To investigate this possibility the scientists set out to immunize atherosclerosis-susceptible mice with different apo B peptides fragments found to activate immune responses in humans. However, giving peptides alone is known to provoke a very weak immune response. To enhance the response they instead constructed a simple vaccine in which aldehyde-modified apo B-100 peptide fragments were linked to albumin in an aluminium salt solution. The large albumin protein serves as a carrier for the small peptides that would otherwise be easily washed away before reaching the proper immune cells and the aluminium salt as an adjuvant that functions as a general activator of immune responses. The combination of an antigen (with a carrier if the antigen is small) and an adjuvant constitutes the bases for all vaccines. Vaccines based on number of different apo B-100 peptide fragments were tested and a handful of peptide vaccines that reduced the development of atherosclerosis by 50-60% identified (figure 5). |
| These findings were encouraging and suggested that it could be possible to develop an atherosclerosis vaccine for human use. However, they also raised the question why the naturally occurring immune responses appeared to fail to provide a sufficient protection against atherosclerosis. An interesting observation was that in humans, as well as in the hypercholesterolemic mice, the immune response was to a large extent mediated by generation of unspecific IgM, whereas immunization with oxidized LDL or apo B-100 peptide fragments shifted the immune response towards the generation of more specific IgG. To investigate if these IgG were directly involved in mediating the protective effect of immunization, human IgG specific for one of the aldehyde-modified apo B-100 peptide fragments was produced by recombinant DNA technology. Repeated treatment of mice with this recombinant anti-apo B-100 peptide IgG reduced atherosclerosis almost to the same extent as vaccination with the corresponding peptide. In contrast, treatment of mice with IgM against oxidized LDL phospholipids failed to reduce atherosclerosis. These findings suggested that activation of adaptive immune responses against structures in oxidized LDL and generation of specific IgG were key mediators of the protective immune response and that the insufficient protection of the natural immune response against oxidized LDL could perhaps be explained by the absence of a shift from innate to adaptive immunity. They also suggested that might be possible to develop a treatment of atherosclerosis based on injection of recombinant oxidized LDL specific IgG. |
| At the same time researchers working in the laboratories of George Wick in Austria and Y Shoenfeld in Israel focused on some other proteins of potential importance for the development of atherosclerosis, the heat shock proteins (Hsp). The Hsp constitute a family of proteins that are expressed in cells exposed to severe stress. The name stems from their initial identification in cells stressed by exposure to increased temperature. The function of the Hsp remains to be fully understood but they appear to protect key cellular proteins from injury in situations of potentially toxic stress. The Hsp in some types of microorganisms, such as Chlamydia pneumoniae has a structure that is almost identical to the Hsp expressed in human cells. There is strong evidence suggesting that if Hsp is expressed in stressed endothelial cells at the same time as immune responses are generated against Hsp in micro-organisms stressed by an attack of the immune system antibodies are produced that will react with Hsp in both cell types. When antibodies bind to Hsp in stressed endothelial cells this promotes vascular inflammation and development of atherosclerosis. Their idea received further support from the observations that immunization of hypercholesterolemic animals (that are known to have increased endothelial stress) with bacterial Hsp resulted in an aggravation of atherosclerosis. Although these immunizations had an adverse effect on atherosclerosis they were still supportive of the idea that it might be possible to develop an atherosclerosis vaccine because vaccines can also be designed to down-regulate ongoing hazardous immune responses. This is generally achieved by a mucosal administration of the vaccine, ie by an oral intake or by an intra-nasal spray. This approach is taking advantage of the fact that the body must develop tolerance towards a large number of foreign antigens present in the food we eat. The next step was therefore to produce an Hsp-based vaccine that could be used for mucosal administration. This vaccine was found to significantly reduce atherosclerosis in experimental animals when |
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