The Indirect Template Theory of Antibody Production (1949) – ViroLIEgy

From 1900 to 1949, there were two main competing theories attempting to explain antibody production and formation as they had never been purified nor isolated directly from the fluids and proven to exist. Antibodies were still unseen fictional creations assumed to be within the blood acting as defenders against invading pathogens, thus researchers needed to take unrelated experimental results produced by different researchers over the years and cram them together in order to fit into a cohesive narrative. The first of these was the Side-Chain theory formulated by Paul Ehrlich in 1900. It is considered part of the Selection theories of antibody production which believes that antigens react to antibodies already existing within the body. It was the main theory explaining what antibodies are and how they form and function for the better part of three decades. However, in 1930 the Direct Template theory was proposed by Fritz Breinl and Felix Haurowitz and later revised by Linus Pauling in 1940. This was considered an Instruction theory as antigens were thought to play a prominent role by serving as a genetic template used to create specific antibodies. These two theories created a bit of a divide between biologically-trained and chemically-trained immunologists as they attempted to explain, according to their backgrounds, how antibodies and the immune system worked.

Fortunately or unfortunately, depending on how you look at it,  Austrailian virologist Frank MacFarlane Burnet determined in 1949 that a third interpretation was warranted in order to rectify some of the loopholes in the earlier theories. His view of the sea of indirect evidence, hypotheses, and theories produced over the decades led him to be in lockstep with Breinl, Haurowitz, and Pauling’s instructionist view. However, there were some key differences which led MacFarlane to propose what became known as the Indirect Template theory in the second edition of his book fittingly titled “The Production of Antibodies.” The book itself is 142 pages long so, needless to say, I will not be posting the entirety of it here. However, we can find a few sources which paraphrase the theory directly from the book to give us an idea of what MacFarlane proposed.

From Immounopedia.org:

The Indirect Template Theory

“(i) To account for the non-antigenicity of body components these were assumed to carry ‘self-markers;” at some point in the antibody-producing sequence a ‘recognition unit’ was postulated to act as a means of detecting material carrying self-markers and deflecting it from the possibility of immune response;

(ii) To account for the persistence of antibody-producing capacity it was postulated that a ‘genocopy’ of the antigenic determinant was incorporated in the genome of the Stem cell concerned, so allowing the indefinite production of descendant antibody-producing cells;

(iii) This incorporation of pattern determinants into the genetic structure of antibody-producing cells provided some basis for the changes in antibody character that may result from secondary antigenic stimuli or simple lapse of time.”

Burnet, F.M & Fenner, F. The Production of Antibodies. Monograph of the Walter and Eliza Hall Institute, Melbourne, 2nd Edition, 1953

The Indirect Template Theory

From this short excerpt, we can see that MacFarlane was attempting to explain how antibodies persist in the blood to provide immunity after the antigen has been cleared out of the body. As he believed that antibodies continued to circulate in the body long after the antigen was gone, this meant that the antigen could not be the template continuing to produce the antibodies one by one by direct contact. This was in opposition to Breinl, Haurowitz, and Pauling’s Direct Template theory which proposed that the antigen goes into the cell and stamps its code onto each antibody after it is produced. Thus, MacFarlane alternately proposed the Indirect Template theory which stated that the antigen incorporates itself into the genetics of the cell in order to produce an endless supply of antibodies.

In 1950, Nature wrote an article which provides us with further insight into Burnet’s Indirect Template theoty. There are some rather interesting admissions made regarding his work:

PRODUCTION OF ANTIBODIES

“The first edition of this essay, published in 1941, has long been rather hard to come by, and the appearance of a second is therefore proportionately more welcome. Since the publication of the first edition, and to a large extent because of it, antibodies have been interesting biologists more and more, in spite of the inroads of antibiotics into their therapeutic uses.

Dr. F. M. Burnet has not changed his ground. Antibody formation is thought to be the consequence of an inherited change in the pattern of synthesis of serum globulin in mesenchymal cells; that is, a change which endures through repeated fissions long after the physical disappearance of the agent which in the first place brought it about. (It makes no important difference to the theory what sort of cell actually makes antibodies, and the authors now give due weight to recent evidence incriminating the plasma cell and the lymphocyte, or its precursor.) According to Burnet’s theory, therefore, antibody-formation is the outcome of an inherited cellular transformation. The ‘chemical’ theory, which in one form or another the authors couple with the names of Mudd, Haurowitz and Pauling, requires the continued presence of antigen during the entire term of antibody formation, the antigen being (crudely) a mould or template for distorting normal into antibody globulin. It is clearly a matter of the utmost technical difficulty to decide whether antibody formation outlives its stimulus or not, and the evidence one way or the other is at present quite inconclusive.

The incompatibility between the chemical and biological’ theories turns solely on the question of whether or not a cellular transformation is entailed. As the authors point out, the mechanism envisaged by the chemical theory may well prove to be correct in principle, though it may not take place at such a late stage in the assembly line of globulin synthesis as is commonly thought. It is essential for Burnet’s theory that the formation of antibodies is subsidized by a cytoplasmic self-reproducing system. Extracellular proteins like serum globulins are the reproductively inert by-products of the synthetic activity of an enzyme system which is undergoing continual replication in the cytoplasm. Antibody is serum globulin formed as the by-product of an enzyme system which, under the impress of antigen, has submitted to a slight inherited change that is in some important ways analogous to that responsible or the formation of adaptive enzymes in bacteria an analogy supported since the publication of the first edition by Hinshelwood’s interpretation for the adaptive process. The sort of molecular distortion envisaged by the chemical theory might be supposed to occur in the Burnet enzyme system itself rather than in the texture of its finished product-an idea which fits easily into the pattern of modern speculation about the nature of self-reproducing systems.

However, the authors would be the first to admit that much of the evidence they bring forward in support of their views is no more than suggestive. The existence of a short phase of exponential expansion of circulating antibody is poor evidence for the existence of a multiplicative antibody-forming system, though it is certainly consistent with such a possibility; and as evidence that antibody formation outlives its antigenic stimulus, the very long-drawn-out immunity that follows infection by some viruses is admittedly clouded by the possibility that a trace infection by virus does indeed persist. But to say this is to say the worst: this is a book which will stimulate or goad every immunologist into thinking afresh about old problems and coming to terms with new ones. Among the matter new to this edition is an ingenious and most stimulating commentary on the important and disturbing fact that embryos do not form antibodies, worked in with a theory of how antibody-forming cells come to distinguish native from foreign organic molecules. Another is the chapter on transplantation immunity, containing the suggestion that the response to the grafting of foreign homologous cells has something in common with the type of sensitivity provoked by tuberculin.

In summary, the immunologist needs no special inducement to read this essay, for he will do so anyway; but it is important that every biologist interested in the problems of cellular heredity and transformation should be aware of the rather direct bearing of antibody formation upon them.”

https://doi.org/10.1038/166204a0

As can be seen from the Nature article, Burnet believed that antibody formation was “thought to be the consequence of an inherited change in the pattern of synthesis of serum globulin in mesenchymal cells.” Oddly enough, it didn’t really matter to his theory which cells actually produced antibodies as it seemed any old cell would do. It was also admitted that the evidence presented for his theory was only suggestive. In other words, as with the theories before it, there was no conclusive evidence proving Burnet’s theory as the correct one. Perhaps this had to do with the continued lack of direct evidence in the form of purified and isolated particles assumed to be antibodies for which the researchers could study and learn from rather than throwing out guesses as to how these unseen entities look and function based on indirect chemistry experiments?

In the abstract for the book by The Journal of Immunology, it is stated that Burnet’s theory is based on the assumption of a self-producing enzyme. The assumption itself comes from logarithmic curves showing an increase in antibody titer. Because of this, it was assumed that “there must be something, somewhere proliferating” which is just another way of saying “we can not observe the antibodies directly so we must assume their presence indirectly:”

“In this second edition of Burnet’s well-known book on the production of antibodies the authors have clarified and extended the views advanced previously. They believe that the first injection of an antigen causes the modification of intracellular enzymes, so that these become adapted to the antigen. Subsequent antigen injections stimulate the replication of the adapted enzyme units formed. The circulating antibodies are considered as partial replicas of the enzyme units, devoid of enzymic activity.

The assumption of self-producing enzyme units is based chiefly on the initial logarithmic increase of the antibody titer. The logarithmic shape of the curve leads the authors to believe that “there must be something, somewhere proliferating”. They assume that the enzymes which are normally involved in the disposal of expendable body cells, become adapted to antigens which are similar to the normal substrates of the same enzymes.”

https://www.jimmunol.org/content/66/4/485

While it should be a red flag that there needed to be not one but various theories for how antibodies supposedly look, form, and function, there were a few issues related to Burnet’s theory that eventually led to it being disregarded. In a 1994 article by American immunologist Melvin Cohn, it is stated that Burnet’s rejection of the Direct Template theory was weak and that his evidence supporting his argument for antibody production long after the antigen was cleared was controversial and unconvincing. Cohn also pointed out that the argument rested on whether one could measure residual antibody secretion by end cells (plasmacytes) after induction had ceased which was beyond the experimental capabilities:

THE WISDOM OF HINDSIGHT

“As the vast majority of immunologists were “laissez-faire” instructionists, Burnet & Fenner’s rejection of Pauling’s direct template theory might have been important, but their argument was weak. They argued that if antigen were a template, then antibody synthesis would cease when antigen was ridded. They then cited evidence that antibody synthesis continued long after any antigen could possibly be present in the animal, but this remained controversial and unconvincing for a simple reason. All theories, on a priori grounds, require that induction of antibody cease when antigen is eliminated. Consequently, their argument rested on whether one could measure residual antibody secretion by end cells (plasmacytes) after induction had ceased, and this had to be beyond the experimental methodology of the time, thereby leaving in its wake a useless polemic.”

doi: 10.1146/annurev.iy.12.040194.000245.

Another strike against Burnet’s theory was that it was later determined that there were antibodies of different specificities with different amino acid sequences:

“This explained specificity and the secondary response but it was abandoned when it was known that antibodies of different specificities had different amino acid sequence in their combining sites.”

http://ecoursesonline.iasri.res.in/mod/page/view.php?id=61813

It was also stated that in nature, there was no need for the antigen to enter the B cell in order to produce antibodies so this apparently also disproved his theory:

“Burnet and Fenner proposed this instructive theory to explain the synthesis of antibody as an adaptive protein. According to this theory, antigen enters into B cell and it binds to its DNA and modifies it and forms this modified DNA, antibodies are produced against antigen and it also specific for it.  But in nature, there is no need for an antigen to enter into B cell and modify DNA for antibody production because soluble antigen can activate B cell by binding to its cell surface receptor BCR.  Because of this reason, this theory also disproved.”

http://biosiva.50webs.org/plasmacell.htm

In the end, after championing it for nearly a decade, Burnet abandoned his Indirect Template theory and eventually proposed what became the “definitive” antibody explanation known as the Clonal Selection theory in 1957.

In Summary:

• At some point in the antibody-producing sequence, a ‘recognition unit’ was postulated to act as a means of detecting material carrying self-markers and deflecting it from the possibility of immune response
• It was postulated that a ‘genocopy’ of the antigenic determinant was incorporated in the genome of the Stem cell concerned, so allowing the indefinite production of descendant antibody-producing cells
• This incorporation of pattern determinants into the genetic structure of antibody-producing cells provided some basis for the changes in antibody character that may result from secondary antigenic stimuli or simple lapse of time
• In other words, this theory was used to explain how antibodies could remain in the blood to provide “immunity” after the antigen was dealt with
• Antibody formation was thought to be the consequence of an inherited change in the pattern of synthesis of serum globulin in mesenchymal cells; that is, a change which endures through repeated fissions long after the physical disappearance of the agent which in the first place brought it about (i.e. antibodies exist long after the antigen disappears)
• According to Burnet’s theory, therefore, antibody-formation is the outcome of an inherited cellular transformation
• It makes no important difference to the theory what sort of cell actually makes antibodies
• It was a matter of the utmost technical difficulty to decide whether antibody formation outlives its stimulus or not, and the evidence one way or the other was quite inconclusive
• The incompatibility between the chemical and biological’ theories turns solely on the question of whether or not a cellular transformation is entailed
• It is essential for Burnet’s theory that the formation of antibodies is subsidized by a cytoplasmic self-reproducing system
• The sort of molecular distortion envisaged by the chemical theory might be supposed to occur in the Burnet enzyme system itself rather than in the texture of its finished product-an idea which fits easily into the pattern of modern speculation about the nature of self-reproducing systems
• The authors would be the first to admit that much of the evidence they bring forward in support of their views is no more than suggestive
• The existence of a short phase of exponential expansion of circulating antibody is poor evidence for the existence of a multiplicative antibody-forming system, though it is certainly consistent with such a possibility
• Burnet also worked in a theory of how antibody-forming cells come to distinguish native from foreign organic molecules
• The assumption of self-producing enzyme units is based chiefly on the initial logarithmic increase of the antibody titer
• The logarithmic shape of the curve led Burnet to believe that “there must be something, somewhere proliferating”
• Burnet assumed that the enzymes which are normally involved in the disposal of expendable body cells, become adapted to antigens which are similar to the normal substrates of the same enzymes
• Burnet & Fenner’s rejection of Pauling’s direct template theory might have been important, but their argument was weak
• They argued that if antigen were a template, then antibody synthesis would cease when antigen was ridded
• They then cited evidence that antibody synthesis continued long after any antigen could possibly be present in the animal, but this remained controversial and unconvincing for a simple reason
• All theories, on a priori grounds, require that induction of antibody cease when antigen is eliminated
• Consequently, their argument rested on whether one could measure residual antibody secretion by end cells (plasmacytes) after induction had ceased, and this had to be beyond the experimental methodology of the time, thereby leaving in its wake a useless polemic

Frank MacFarlane Burnet’s Indirect Template theory was a short-lived explanation for the production and formation of antibodies that never really got off the ground. It joined two other attempts at taking unrelated indirect experimental findings and forcing them together into a cohesive description for unobserved phenomena. As with the Side-Chain and Direct Template theories before it, Burnet’s Indirect Template theory had its own view and interpretation of the experimental evidence accumulated over the decades. He was able to furnish his own findings as well as those of others in order to support his overall hypothesis. Much like Linus Pauling’s work with the Direct Template theory, Burnet’s proposal was considered a legitimate theory initially. Other researchers built off of his theory with their own work and experimentations. However, Burnet’s original theory eventually fell out of favor which means much time and effort was wasted on a false theory which produced research which could only lead to results which would be considered false and erroneous. The theory led researchers in the wrong direction.

Burnet’s Indirect Template theory is a shining example as to why definitive conclusions should not be made about the assumed existence, form, and function of invisible entities. It is only a matter of time before someone else comes along and interprets the indirect experimental evidence in a different light and proposes another theory which may be accepted by the consensus majority instead. This leads to a revolving door of theories for something which can not be seen nor explained as antibodies started off as an idea rather than an observed phenomena in nature. A theory is supposed to be born out of the observation of natural phenoma, not built from an idea of something invisible assumed to exist within the blood. A theory is supposed to be assembled from facts and results aquired from the implementation of the scientific method. The scientific method requires a valid independent variable in order to determine cause and effect. As antibodies have never been purified and isolated directly from the fluids, there is no independent variable in order to complete the scientific method. Thus the antibody theories are not scientific.

What this means is that there was a massive amount of wasted time and literature that was spent on unscientific and disproven theories such as the Direct and Indirect Template theories. In many cases, this would be fine as this is how science is supposed to work. Theories are made to be disproven and replaced. However, this becomes a problem when unproven theories are used as a means of implementing health measures. Antibodies are presented as if they exist and that the current theory is considered to be the stone cold truth. Medical decisions, treatments, vaccinations, therapies, etc. are made based on the latest favorable theory which may some day soon fall out of favor for something “better.” Decisions are determined during supposed pandemics based on whether or not antibodies exist and function in the way that the theory proposes that they do. However, the evidence is contradictory and the theory remains unproven.

What happens when the antibody theory is ultimately disproven? Where does that leave the person who was vaccinated and believed a theoretical rise in antibodies was a good thing yet ended up injured due to the toxic side effects? How does this help the person suffering serum sickness after taking experimental monoclonal antibody therapy as a cure for a “virus?” What does this mean for the research promising medical marvels and insights which is ultimately unreproducible and irreplicable due to the non-specific reactions of the theoretical antibodies? Where does it leave us when antibodies are used as a means to determine one is “protected” or not in order to give or take away our rights?

Unproven theories are being presented as the unbridled truth and are used to impact our health and our freedoms. Antibodies are the perfect scapegoat to keep us blind to the “virus” lie and believing in the power of vaccination. They are the opposite side of the viroLIEgy coin and the lack of evidence for these theoretical entities must be scrutinized to the same extent.