Conclusions and Summary
The studies discussed in this report have demonstrated the efficacy of a group of branched-chain, saturated, noncyclic hydrocarbons as adjuvants in emulsified polio and influenza vaccines and have elucidated the properties of the compounds required or desirable for their application as antigen potentiators.
Straight-chain hydrocarbons were shown in these studies to be unsatisfactory for use in emulsified vaccines because of their irritating properties. Although this toxic effect diminishes as the carbon number is increased, the melting point of the compounds rises with increasing chain length such that the alkanes approaching suitable innocuity are solid at room temperature.
Branching of the hydrocarbon compounds by introduction of one or more alkyl side chains largely eliminates dependence of the melting point on carbon number, whereas the toxicity and reactivity continues to decline with rising molecular weight and reaches a minimum at approximately 24 carbon atoms, thereby rendering branched-chain hydrocarbons of this number and above most suitable for use individually as the oil continuous phase of emulsified vaccines, at least from the standpoint of innocuity.
The viscosity of the branched-chain hydrocarbons is determined primarily by their carbon number or molecular weight and only secondarily by the position or structure of the side chain. Multiple branching, however, may result in a marked drop in the melting point and a substantial rise in the viscosity of the hydrocarbon.
The viscosity of hydrocarbon-emulsified vaccines is closely correlated with the viscosity of the corresponding hydrocarbon compound itself, the relationship being expressed by the following empirical equation: log η = K + η0 Ø3
η = vaccine emulsion viscosity in centipoises;
η0 = hydrocarbon viscosity in centipoises;
Ø = volume fraction of the disperse phase;
K = a constant, equal to 1.89 for emulsified vaccines containing 7.5% Arlacel A emulsifier (final concentration).
Increase in temperature reduces vaccine emulsion viscosity, the extent of reduction apparently varying with the log of the original emulsion viscosity. At human body temperature, however, the residual viscosity of emulsified vaccines is considerably above that of the body fluids and presumably will assist in retention of the vaccine at the injection site.
Potency evaluation in rabbits of type 1 polio vaccine emulsified with ten representative branched-chain hydrocarbons indicates that approximately maximal levels of antibody are achieved within 2 weeks after inoculation, the high levels being maintained for as much as 22 weeks longer. The hydrocarbon-emulsified vaccines, moreover, produced a 3- to 16-fold greater antibody response than was achieved with the fluid vaccine alone; and with the exception of one of the two most viscous compounds studied, all of the hydrocarbons were superior to Drakeol 6VR oil as adjuvants, 6 of the 10 compounds evoking at least twice the response of the mineral oil.
Antibody response to the emulsified vaccines was found to be correlated with both vaccine viscosity and irritating capacity of the hydrocarbon and to a closely similar extent. The direction of potency correlation, however, differed with the two factors, response varying directly with irritability and inversely with vaccine (and hence, hydrocarbon) viscosity.
Studies on the combination of hydrocarbons demonstrate the possibility of increasing the adjuvant activity of the higher molecular weight compounds without raising their toxicity to objectionable levels by the addition of various amounts of the shorter chain, toxic hydrocarbons. Combination of 40 to 60% 7-n-propyltridecane in 7-n-hexyloctadecane more than doubles the adjuvant activity of the latter hydrocarbon. Polio vaccine emulsified with the 40% hydrocarbon combination possesses 22 times the potency of the nonemulsified fluid preparation and 5 times that of emulsified Drakeol mineral oil vaccine. Similarly, 60% 2-methyl-3-isopropyl-tridecane in 7-n-hexyloctadecane raises the adjuvant activity of the latter compound almost 2-fold. Although hydrocarbon toxicity and the corresponding log vaccine viscosity show a linear relationship with increasing concentration of the shorter chain component of combined hydrocarbons, maximum vaccine potencies were achieved with combinations short of 100% of the lower weight compounds, indicating that factors other than viscosity and irritating capacity are responsible for hydrocarbon adjuvant activity. Cytopathology studies have shown a difference in the cell type attracted to the injection site following inoculation of vaccines emulsified with irritating and innocuous hydrocarbons, suggesting this factor as responsible for differences in the pattern of antibody response and for the higher titers obtained with combined hydrocarbons.
Studies on influenza (PR-8) vaccine emulsified with three representative hydrocarbons have confirmed the efficacy of these compounds as vaccine potentiators when determined by protection against direct virus challenge as well as by antibody response, the case with poliovirus vaccine. With the shortest chain hydrocarbon tested (7-n-propyltridecane, C16) the peak response to emulsified influenza vaccine was higher and occurred earlier than with the heavier C24 compound (7-n-hexyloctadecane), 3 weeks compared with 6 for the latter. After the time of maximal response, protection provided by the lower weight compound declined to the peak level of the heavier C24 compound which was maintained for a period of at least 24 weeks, the time the test was terminated. As in the case with polio, adjuvant activity of the hydrocarbons in influenza vaccine was inversely related to carbon number, although all three of the hydrocarbon compounds tested produced higher and longer lasting levels of protection than did the nonemulsified fluid vaccine (2 to 560 times the protection and at least 6 to 12 weeks longer).
The results of these studies on 10 representative hydrocarbons with 2 virus agents have demonstrated the nature and extent of the vaccine potentiating capabilities of this class of compounds. Their efficiency as adjuvants recommends exploration of their potential in virus and in bacterial vaccines for human and veterinary use.