Antigen-Antibody Interactions in vitro: Analyses Demonstrate a Dominating Role of the Non-Neutralizing Antibodies as the Most Potent Inactivators of Viruses

The understanding of the regular lines of reactions between virus and antibodies has been hampered by the common perceptions: 1) that neutralization of virus in neutralization tests would lead to a state of equilibrium and 2) that it was generally thought that non-neutralizing antibodies did not participate in the neutralization in vitro. The regular lines of neutralization by antibodies in vitro have been presented earlier, and in a recent article, the reaction characteristics in five different assays demonstrating antibodies to viruses were analyzed and recommendations for gold and reference standard tests and routine adequately sensitive tests were given. Inactivation of the virus in a conventional neutralization test was found to be a bifactorial process, consisting of: 1) a prompt and short-lasting reaction originally termed the over-neutralization phenomenon and 2) an enduring but slowly progressing reaction of first order. The over-neutralization could later be concluded to be neutralization due to the aggregation of viruses. The reaction of first order followed the lines of a demonstrated formula for the regular antigen-antibody interactions in vitro, not including aggregation. Tests for demonstration of viruses and antibodies of very high sensitivity and specificity have been elaborated on the basis of these reaction lines and used in diagnosing, controlling and eradicating viral infections in the veterinary medical field for a long time.

In the present article, the role of the non-neutralizing antibodies in the neutralization process in vitro is analyzed. The non-neutralizing antibodies inactivate viruses by aggregating the agents. An extremely high virus-inactivating potency of these antibodies was demonstrated. The principal characteristics of virus aggregation were concluded to be the following: 1) IgG and IgM antibodies to various antigenic determinants aggregate virions synergistically and rapidly, 2) the virus-inactivating aggregation by the polymerized isotypes of antibodies is greatly enlarged by their polyvalency, and 3) the complement component C1q will promptly attach to antibodies sensitized by being bound to their antigenic determinant on a virus and inactivate such virus-antibody complexes by including them in aggregates. In complement-enriched neutralization tests, C1q will 1) promptly aggregate antigen-antibody complexes formed and 2) with increasing reaction times aggregate the virus following the first-order binding of non-neutralizing antibodies to their antigenic determinants, thereby inactivating viruses with the same rate as neutralizing antibodies.

In a herpesvirus complement-enriched neutralization test, the titers of reacting non-neutralizing IgG antibodies in the highest concentration were found to be approximately 8 times higher than that of the neutralizing antibodies, indicating that also the concentration of the reacting non-neutralizing antibodies was 8 times higher than that of the neutralizing ones. The total concentration in blood of non-neutralizing antibodies in blood by far exceeds that of the neutralizing ones, theoretically being largely proportional to the number of antigenic determinants on a virion. The capability of the non-neutralizing antibodies to neutralize by aggregation, most pronounced in cooperation with the C1q complement component, and their high concentration, imply that the non-neutralizing antibodies have a much greater neutralizing potency than neutralizing antibodies. One non-neutralizing antibody bound to an antigenic determinant will result in almost immediate inactivation of the virus due to the incorporation of the formed antigen-antibody complex in aggregates by the C1q component.

The formation of the pentameric and decavalent IgM antibodies is the first humoral immune response after infection and the virus-inactivating potential of these acute-infection phase antibodies is enormous. A significant neutralizing IgM antibody response could be detected in blood by a complement-enriched neutralization test 4 days after nasal infection and samples taken after 8 to 15 days were positive in dilutions 1:10.000 or higher. The IgM antibodies, non-neutralizing as well as neutralizing, must in cooperation with the C1q component of complement be considered the ultimate virus inactivators and of almost unimaginable importance in the defeat of infections.

Recognized but unidentified specific forces attracting: 1) the binding sites of antibodies and their specific determinants on viruses, and 2) the binding sites of the C1q component and the Fc fragment of sensitized antibodies bound to their antigenic determinants, are concluded to be one reason for the prompt reaction, which is characteristic of the virus aggregation process.

Further investigations into the effector mechanisms determining: 1) the specificity and attractive binding of the antibodies to their antigenic determinants and 2) the reaction between the complement component C1q and sensitized antibodies are urgently needed.