Melvin Cohn and Rodney E. Langman

The Salk Institute, P.O. Box 85800, San Diego, California, 92186-5800

Received 8/15/96; Accepted 9/9/96; On-line 10/1/96

5. THE ESSENCE OF THE HUMORAL RESPONSE, THE CONCEPT OF A PROTECTON

What activities constitute the evolutionary selection pressure that shaped the humoral response?

The analysis of this question leads to a new concept that will appear at first somewhat strange. Humoral antibody effector functions depend upon secreted immunoglobulin. The concentration of antibody must reach a minimum effective threshold in a short enough time to stop a growing pathogen before it becomes lethal. This requires that initially an equivalent number of B-cells per ml respond to the pathogen. This number of B-cells must respond for each and every milliliter of animal. Consequently, the humoral immune system must be iterated. This straightforward conclusion has far reaching implications.

Before discussing the implications, let us give some rough numbers that would illustrate this concept. First, is that a threshold antibody concentration of 100ng/ml must be reached within 5 days to protect against the 'worst case' pathogen. This would require that roughly 200 B-cells per ml specific for the pathogen be present initially. This applies to each and every milliliter of animal. Second, the iterated unit must be sufficiently diverse to be protective against a variety of pathogens - missing, say, 3 in every 10³ pathogens. Third, there is a limit to the total number of B cells per ml that is around 10⁷/ml for most species. The iterated unit, then, must have a minimum total size and a concentration parameter. Our best estimate is that the iterated unit is a total of 10⁷ B-cells, at a concentration of 10⁷ B-cells per ml with roughly 200 B-cells per ml responsive per pathogen. We refer to this iterated unit of protection as a Protecton. The Protecton is the target of evolutionary selection on the humoral immune system.

Consider a pygmy shrew with 10⁷ total B-cells, a mouse with 10⁸ B-cells, a human with 10¹² B-cells and an elephant with 10¹⁴ B-cells. This translates into a pygmy shrew with 1 Protecton, a mouse with 10 Protectons, a human with 10⁵ Protectons and an elephant with 10⁷ Protectons. These animals are equally protected against their pathogenic universes by their humoral immune systems. They are protected per milliliter not per animal. All Protectons are equivalent in function.

There are four points to make before confronting several implications of the concept of a Protecton.

1) The Protecton is defined as the smallest sample of the humoral immune system that retains all of the evolutionary selectable protective properties of the whole.

2) While, for simplicity, we have treated Protectons as independent units, there is a factor of cooperativity between them, but this is second order for our discussion.

3) The minimum Protecton unit we have introduced is based on protection against a 'worst-case' bacterial infection. On the one hand, there are some exceptionally lethal pathogens, especially those producing potent toxins, that are largely beyond immune control. These exceptionally fast growing virulent pathogens are rarely encountered and must be self-limiting for non immune reasons_otherwise there would be no surviving vertebrates with immune systems. On the other hand, there are pathogens that grow relatively slowly but require rare and highly specific antibodies to limit their growth. A Protecton can be defined on the basis of the immune response to a particular infection and in the case of a slow growing pathogen it could take as long as 10 or even 20 days before the threshold concentration is reached. A Protecton for a slower growing pathogen might be 106 cells or for a faster growing pathogen, closer to 10⁸ cells. Thus, a Protecton is a vectorial quantity that is directed against each individual pathogen as it is encountered.

4) The Protecton characterizes the primary encounter with a pathogen. This is the key step in evolutionary selection. If the response of the virgin immune system cannot protect against a pathogen then the response on secondary encounter would be of little interest. The secondary response is essentially a byproduct of an effective primary response, the direct target of evolutionary selection.

5.1. Some consequences of Protecton theory

Now let us look at the consequences of this concept that the humoral immune system is iterated.

Immunologists have always viewed the immune system as being able to call upon a transcendental repertoire for an effective response. The early models of diversification might best be described as "big bang." The repertoire was viewed as being expressed in its totality in one step whether this step was the combinatorial expression of many germline V-genes segments or this step was the hyper recombined V-gene segments or of hyper varied V-gene segments by random replacement by minigenes of their complementarity-determining regions. While big-bang seemed to describe the observations it lacked any credible arguments of evolutionary necessity. We have argued that the repertoire must be expressed in two stages. STAGE I is a small germline encoded repertoire that is represented in high copy number, and also acts as a substrate for STAGE II, which is generated by somatic diversification (hyper mutation) and is in single copy. This view has met with strong resistance largely because it was derived as an evolutionary necessity not as a direct observation.

When it was learned that a relatively small number of V-gene segments were present in the genome, big-bang fell briefly from favor. A short time later the "big bang" model was reborn like the Phoenix when junctional diversity and an extra D-gene segment was discovered. This allowed enormous repertoires to be derived by multiplication of numbers of rearranging gene segments by functional joining variation by subunit complementation to arrive at repertoire sizes in excess of 10¹⁰. Every review and textbook covering repertoires carries this calculation and the term 'complete' has become popular to describe the range of these repertoires.

The concept of a Protecton has made this calculation misleading. Clearly the size of the available repertoire cannot be larger than the number of B-cells per Protecton, that is, 10⁷. To illustrate, consider a mouse with 10⁸ total B-cells. If the repertoire were 10¹⁰ (the usual minimum estimate) and any one of those specificities were important for the protection of an individual, then only one in 100 mice would express that specificity at any given moment in time and even that mouse would be unprotected unless it was allowed to take almost 30 days for the one B-cell to multiply to a protective level. The latter would even be true for a human with 10¹² total B-cells. The individual would express 10² total B-cells specific for the pathogen but being too few they would respond too slowly to protect. Vast, transcendental repertoires are evolutionarily unselectable as such because they are of a nonfunctional size.

Returning now to a more realistic estimate of the size of the functional (or available) repertoire, an upper limit in principle is 10⁷ based on 10⁷ B-cells/ml; but, this too is a substantial overestimate. An analysis of the pathway of expression of the Protecton places the repertoire at about 5x10⁴. This repertoire is composed of a germline (STAGE I) repertoire of ~10⁴, but each specificity is present in high copy number (~10² B-cells per specificity per Protecton) and a STAGE II somatic mutationally derived repertoire of ~4x10⁴, which is in low copy number (1 B-cell per specificity per Protecton). These two repertoires interact synergistically to provide a sufficiently rapid response to a large enough family of pathogens.

5.2. The primary repertoire and the pathogenic universe

As a rough estimate, this virgin repertoire protects the individual at the 99% level. This is the limit to evolutionary selection because other factors such as the probability of being eaten by a predator or of starving becomes the limiting factors for survival. What the immune system really does is seen in immune deprived individuals where a surprisingly large family of pathogens are revealed as 'opportunistic'.

Protecton theory highlights a detail of effector function that is very important for the design of vaccines and passive antibody treatment. A monoclonal antibody may neutralize a pathogen or toxin by blocking attachment to its target or by inactivating an enzymatic activity, but it is ineffective in ridding the antigen. Ridding is largely a function of opsonization by macrophages and this requires the formation of a three dimensional aggregate of antibody.

By way of illustration, consider a monomeric antigen like diphtheria, tetanus or cholera toxins. A monoclonal antibody might neutralize its toxicity, but because it cannot form a aggregate with the antigen, the toxin would not be effectively ridded. Two monoclonal antibodies reacting with different determinants on the monomers would form a linear chains of immunoglobulin, and that too is inefficiently opsonized. It takes 3 or more antibodies reacting with different determinants to form the three dimensional aggregate that is ridded efficiently. Neutralization does play an important role by giving the immune system more time to respond and produce the ridding antibodies.

Because evolution selects on the limiting case, on average three or more antibodies would be induced by polymers even though a monoclonal antibody reacting with a polymer might be sufficient to allow aggregation. However, antibody aggregated on a virion is less effective in ridding the virus than virions aggregated by antibody. Whether a monoclonal antibody interacting with virions will cross link or bivalently bind depends on the spacing of the ligand recognized. If 3 or more antibodies bind, cross linking is assured and ridding is effective.

The repertoire of ~5x10⁴ specificities in the Protecton divides the antigenic universe into epitopes distributed randomly and combinatorially on antigens, ten at a time. The total number of antigens distinguishable by this repertoire is ~10⁴³ (5x10⁴C10), a big enough number for any theory. This repertoire will "miss" 3 in 1000 antigens because they will be seen in less than 3 ways by the Protecton. The number 10 epitopes per antigen is an estimate based on a computer modeling study of the Protecton.

It might seem surprising that a small repertoire can deal with a large antigenic universe. An understanding of how this works begins with four points.

1) It is the paratope (combining site) that is primary as the target of evolutionary selection. Only paratopes, define epitopes.

2) A given paratope (antibody) that reacts with several epitopes distinguishable by the immunologist (referred to as crossreactivity) treats these epitopes as a single epitope functionally. Any antibody that recognizes an epitope present on a self and a nonself antigen is defined by the immune system as anti-self and the epitope as a self-epitope.

3) Symmetrically, if a given epitope is recognized by several paratopes (antibodies) distinguishable by the immunologist (referred to as degeneracy), the immune system treats this family of antibodies as one antibody functionally.

4) The paratope looks at "shape" and a given paratope-defined shape can be created from many different chemical structures. For example, an anti-carbohydrate paratope can be found to react with a peptide. It is this recognition of shape, not chemistry, that allows the paratopic repertoire to divide the universe of chemically different antigens into a limited number of epitopes. Paratopes define antigens as collections of linked epitopes combinatorially distributed.