The Relation of Antibody Response in Swine to Dose of the Swine Influenza Virus Inactivated with Formalin and with Ultraviolet Light

Abstract

Discussion and Summary The results of the present work show clearly that the degree of antibody response of swine is related to the dose per unit weight of formalin- or ultraviolet light-inactivated swine-influenza virus. Within the limits of the range of doses employed, 0.1 to 5.0 mg per 100 1b, the relation of log antibody titer to log dose of vaccine per unit weight appeared linear. With a single vaccination with formolized virus, a 10-fold difference in dose resulted in a 2.5-fold difference in antibody titer; a similar difference in the dose of ultraviolet vaccine was associated with a 1.6-fold difference in titer. The analogous relations for the second vaccination were a 1.6-fold difference in titer with a 10-fold difference in the dose of formalin vaccine and a 1.7-fold difference in titer with a 10-fold difference in the dose of ultraviolet light vaccine. The results likewise show clearly that the influence of dose on the peak level of antibody titer within the range of doses studied is relatively small in comparison with factors associated with the immunological status of the host with respect to experience with the influenza virus prior to the time of vaccination. Whereas at the first vaccination the difference between the log titers of the smallest dose of formalin vaccine, 0.1 mg per 100 lb, and the largest, 5.0 mg per 100 lb (a 50-fold range in dose) was log 0.67 (4.6-fold range of titer), the difference between the log titer associated with the dose of 0.1 mg per 100 lb at the first vaccination, log 1.96, and that following a like dose at the second vaccination, log 2.84, was log 0.88 (7.6-fold range in titer). From inspection of the results of a single vaccination (figure 8) it might appear that doses of vaccine in the range studied called forth responses which were near the limit of reaction possible for the host. That this is not the case is seen from the results with like doses of vaccine at the second vaccination (figure 13), and the results obtained with the animal, No. 65 (figure 22), which possessed-demonstrable antibodies before any vaccination. It is thus evident that while the magnitude of dose is a factor of relative influence on the antibody level under given conditions, the ultimate limits of the capacity of the host to respond are determined to a greater extent by immunological mechanisms inherent in the host itself. These findings are by no means unique; instead, the results conform closely to the generality of experience relative to host response to other antigenic materials. In so far as the character and the scope of the experiments are comparable, the findings of Hirst, Rickard, Whitman and Horsfall (2) in studies in man are similar to those with swine. These authors found that a 100-fold range in dose of influenza virus A vaccine was associated with a 2.5-fold range of antibody response (table 2 of (2)). In the instance of influenza virus B vaccine, there was a 2.4-fold range of response corresponding with a 40-fold range of dose. These results with man are quantitatively comparable with the present findings in swine. It should be noted, however, that the interpretation by Hirst, Rickard, Whitman and Horsfall “that the average antibody response of human beings is directly related, though not strictly proportional, to the amount of virus given” is somewhat of an overstatement in view of the quantitative relations actually found. Further parallelisms between the behavior of swine and man are the rapid loss of antibodies after vaccination and the more rapid decline in titer in those individuals in which the peak of titer was high than in those exhibiting the lower titers. The significance of the results of the studies on dose-response in swine is of chief interest in the possible bearings of the findings on the problem of vaccination of man for protection against infection with the influenza virus. With access to preparations of highly concentrated and partially purified virus, it is obviously desirable to consider the use of such material for preparing vaccines. Especially attractive in this respect is the possibility of grading the dose of vaccine to optimal proportions relative to 1) response; 2) the toxicity of the vaccine; and 3) the cost per dose of producing the vaccine. The range of dosage used in the experiments with swine, 0.125 to 2.0 mg per individual, was based on practical consideration. The quantity of 2.0 mg of virus per ml was attained by a concentration of the virus to a degree approximately 100 times that present in the chorio-allantoic fluid. A volume of 100 ml of chorio-allantoic fluid is approximately that contained in 10 to 11 eggs at the time of harvest, 42 hours after inoculation with the swine virus. With consideration of the technical procedures and materials, including eggs, necessary for producing large quantities of the vaccine, this amount, 2.0 mg per ml, seemed about the maximum desirable. The smallest dose, 0.125 mg of virus (vaccine), represented the quantity that could be recovered from about 6.2 ml of chorioallantoic fluid and thus lay within the range attainable by other methods which have already been extensively used for concentration, as, for example, the adsorption on and elution from chicken red blood cells. The yield of influenza virus B (Lee strain) is about 5 mg per 100 ml of chorio-allantoic fluid infected with this strain (13) and of influenza virus A (PR8 strain) 5 to 10 mg for a like volume (13). Thus the smallest dose of swine-virus vaccine, 0.125 mg, likewise corresponds to an amount of the human types of virus which can be obtained from a single egg and concentrated into a volume of 1 ml. From the point of view of yield and cost of virus and the difficulty associated with the technical procedures of virus concentration, the range of swine-virus vaccine, 0.125 to 2.0 mg, corresponded to a range of influenza-virus B (Lee strain) vaccine of 0.3 to 5.0 mg and of influenza-virus A (PR8 strain) vaccine of 0.3–0.6 to 5.0–10 mg. The range of swine-virus vaccine dose employed in the present work, therefore, parallels a range of doses of human vaccine varying from quantities technically simple and relatively inexpensive to obtain to amounts bordering on the limits of accessibility from a practical point of view. A factor of considerable importance in the choice of dose is the toxicity of the vaccine, that is, the general reaction of the host to administered vaccine aside from that concerned with the infective properties of the agent. In swine the largest dose of formolized vaccine per individual tolerated without evident reaction was 0.5 mg. This is likewise approximately, or perhaps slightly more than, the amount which has been administered on a large scale to man (5, 6). This statement is based on the consideration that if influenza A or B virus-infected chorioallantoic fluid contains 0.05 mg per ml of the respective viruses, a quantitatively 10-fold concentration by red blood cell adsorption and elution would yield 0.5 mg of virus per 1.0 ml of the concentrate. In the final choice of dose, it would appear desirable to employ the largest quantity of vaccine compatible with the various factors. From the point of view of toxicity, the dose 0.5 mg is within the limits of host tolerance. This quantity can be obtained simply and relatively inexpensively in a volume of 1.0 ml and is of a size which would permit the inclusion in a single inoculum of a mixture of the two types of virus (A and B) in quantities of 0.25 mg each. It would be possible also to employ mixtures of various strains of the respective types currently responsible for infection in the general population. The above discussion has been concerned for the most part with dosage, that is, with a consideration of the amount of vaccine that would be introduced at a single injection under any given set of conditions. Another problem, which is actually of greater importance from the point of view of practical protective vaccination, is concerned with the manner or sequence of applying the optimal dose. The experiments with swine show that, irrespective of the dose, a single vaccination is relatively ineffective and inefficient in the induction of antibodies in comparison with the effects of repeated vaccination. Furthermore, as shown both in swine and in man, the effects of either 1 or 2 vaccinations are shortlived. In man, inasmuch as antibodies are nearly universally present, it can be considered that a single vaccination would be equivalent, essentially, to a second vaccination in a host that had not had previous infection or contact with the virus. Such a concept, while adaptable to the conditions at the moment, fails to take into account the possibilities of future years. The hopes of success of a practice of a single vaccination under this concept would be largely dependent on the necessity for previous infection or contact of the host with the infectious virus in nature. Moreover, it would be necessary either to assume that the time relations between previous infection or contact and the moment of vaccination were uniform within the population or discount to a considerable extent the possible influence exerted by this relation. It must be recognized, of course, that the degree of protection against subsequent infection afforded either by previous infection or by vaccination under any conditions yet employed is relatively not great. Yet this knowledge cannot be permitted to interfere with the application of optimum immunological principles in the attempt to lessen the frequency of infection with influenza-virus. From this point of view it is evident that, if resistance to infection is related or proportional to, though not necessarily dependent on, the degree of antibody response and to the longevity of the antibodies induced, then multiple administrations of vaccine, even in minute quantities, would be expected to be of far greater importance than the magnitude of the dose to be given at one time. The question of the optimal frequency of vaccination is still to be investigated. The experiments with swine show that the antibodies arising as the result of a single vaccination decline to a low level by the end of the 3-weeks' post vaccination period. In contrast, the level of antibodies attained after a second vaccination was still high a month later and no lower after 6 weeks than the antibody level reached in the decline in 3 weeks following the first vaccination. Regardless of whether resort will be had finally to multiple injections of vaccine throughout the influenza season, it is clear from the results already at hand that repetition of vaccination provides a far greater promise of maintaining a high antibody level through the influenza season than the application of a single injection of the largest dose of vaccine thus far investigated.