antibody response of fish to viral antigens

ANTIBODY RESPONSE O F FISH TO VIRAL ANTIGENS*

M. M. Sigel and L. W. Clem Department of Microbiology, Universi ty of Miami School of Medicine,

Coral Gables, Flu., and Lerner Marine Laboratwy , B h i n i , B. W . I .

INTRODUCTION It was previously reported from this laboratory that marine fishes can

make antibodies to PR8 influenza virus.I , 2 The initial studies have now been extended to other viruses. These, along with further observations on the nature of the immunologic response, and on quantitative and qualitative differences in the response of fishes belonging to four families of two classes, are the subject of the present paper.

MATERIALS AND METHODS Animals . The marine fishes were caught in the waters surrounding

Bimini island and were maintained in pens and floating cages constructed in the bay near the docks of the Lerner Marine Laboratory. They included two species of elasmobranchs, the lemon shark (Negapr ion brevirostris- P o e y ) , the nurse shark (Ginglymostomn cirratum-Bonruterre) , and one teleost, the margate (Haemzclon album.-Cztvier). Most of the lemon sharks weighed from 18 to 24 kg., average 21 kg. ; the body weight of the margates ranged from 600 to 800 gm. Several newborn nurse sharks were also used and these weighed approximately 100 gm. The baby sharks were heid in plastic tanks supplied with running sea water. The amount of antigen was the same for all animals of a given weight group and the results indicated that the variation in weight of animals of a group did not significantly affect the immunologic response.

The water in which the animals were maintained is of high clarity and its temperature ranges from 27 to 30°C. The losses among these animals were minimal and many have survived for periods of 6 to 10 months.

The gars, fresh water holostean fish (Lepisosteus platyrhincus-DeKay) came from the Florida Everglades. They weighed 500 to 800 gm. and were housed in a fresh water cement pond of a local fish hatchery.

Injections and bleedings. The animals were anesthetized with MS 222- Sandoz (tricaine methanesulfonate) in concentrations of 1 :4000. The an- esthetic in sea water was sometimes administered with a bulb syringe into the mouth and region of the gills, or, which proved most convenient, was placed in a bucket or wash tub and the fish were allowed to swim in it.

*This investigation was supported by U. S. Public Health Service Research Grant No. A1 05758 from the National Institute of Allergy and Infectious Dis- eases and U. S. Public Health Service General Research Support Grant No. F R 0536 from the National Institutes of Health. Some aspects of the field studies were aided by funds provided by Contract No. NONR 552 (07) between the Office of Naval Research and the Lerner Marine Laboratory.

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Sigel & Clem: Antibody Response 663

The fish stopped moving in one to five minutes and could be handled with great ease. Sharks were bled from the hemal a rch ; the needle puncture was made through the ventral midline posterior to the cloaca. Blood of margates and garfish was obtained by cardiac puncture. Except where otherwise noted the injections were made underneath the skin. Because of the t ight attachment of skin to muscle, this subcutaneous mode of injection most probably resulted in the deposition of considerable amounts of antigen in the muscular tissues. Formation of a bleb at site of inoculation was easily discernible in the sharks but not in the margates.

HemaggEutination inhibition ( H I ) test. The test was performed by the well-established technique utilizing twofold dilutions of serum (0.2 ml.) , four hemagglutinating units of virus (0.2 ml. ) and one per cent suspension of guinea pig RBC (0.2 ml.). The sera were heated a t 56" for 30 minutes and treated with a concentrated suspension (10 per cent) of erythrocytes in order to remove naturally occurring hemagglutinins.

Other procedures and tests will be cited under specific experiments when pertinent.

Viruses. The following viruses were used: PR8, human influenza type A ; AZ/Equi/Miami/63, the prototype strain of North American equine influenza type A ; 3 and Sendai, mouse parainfluenza virus.

EXPERIMENTAL Pattern o f Immunologic Response o f Sharks

The production of H I antibodies by lemon sharks to myxoviruses is pictured in FIGURES 1 to 3. FIGURE la illustrates the responses of four sharks to the PR8 virus administered in 4 ml. doses (divided among four subcutaneous sites). The initial series of immunization consisted of five weekly injections and the quantity of antigen corresponded to 5,000 to 10,000 hemagglutinating (HA) units per ml. The t i ters rose slowly and reached a peak a t 30 days, one week after the fourth injection. Following this there was a decline of antibody production despite the additional inoculation at 30 days. What appears t o be of special interest is the absence of fur ther increase in t i t e r even with a booster inoculation given on day 100 to the one animal which has been studied over an extended period of time. This failure to elicit an anamnestic response during the first three months also has been observed in other sharks and other fishes, as will be shown subsequently. Increases in antibody levels were, however, obtained when fur ther antigenic stimulation was provided on day 151 and 253, but these were not of an enhanced type. The kinetics of the response and the level of antibodies demonstrable in the serum appeared to be similar to those obtained during the initial course of immunization.

The production of HI antibodies was not dependent on administration of live virus; as shown in FIGURE l b , formalinized PR8 virus also elicited

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antibody formation. These two sharks were injected at weekly intervals with 4 ml. of virus which had been treated with formalin in 1:4000 dilu- tion. This preparation contained no viable virus as determined by inoculation to chick embryos. The rate of antibody production appeared to be similar to that obtained with live virus but the peak levels were lower: range of 1 :160 to 1 :1280 in the present group versus 1280 to 1 :20,480 in the live vaccine group. The t i ters began t o decline following the 40th day bleeding. On the 87th day these animals were injected with 4 ml. of live PR8 virus, but failed to manifest an increase in t i ter when tested on the 91st and 105th day.


In FIGURE l c are shown the curves of antibody responses of two sharks to the equine influenza virus. The responses were not unlike the ones to the human influenza virus, and the peaks were reached between 28 to 40 days. Thereafter the antibodies began to diminish and again there was no increase to an inoculation given a t 75 days. The maximum titers were lower than those which developed in response to PR8 virus. I t is possible that this difference was a reflection of a dose effect (observed previously in the immunization of lemon sharks) since the antigen content as measured by the amount of HA was at 10 times smaller in the equine virus preparation than in the PR8 virus (approximately 600 HA units/ml. versus 5,000 to 10,000 HA units/ml.). In experiments not presently reported in which parainfluenza type 2 virus was used as antigen, no immunologic response could be elicited when antigens with a hemagglutinating potency of approximately 100 hemagglutinating units were used. However, until more information becomes available on the minimal effective doses, other factors cannot be ruled out as possible determinants of the difference in the responses to PR8 and the equine influenza viruses.

A similar immunologic pattern was noted in sharks immunized with the murine parainfluenza virus, Sendai, as shown in FIGURE 2. The maximum antibody level was attained between 28 and 35 days of the first injection. This level was maintained for a t least 20 days but when next measured af ter 30 additional days the titers had dipped significantly. Again the antibody level could not be elevated by an additional injection, in this group given on the 88th day, but in the one animal an injection administered on the 143rd day did promote a booster effect. Once more the effect was not truIy anamnestic in nature - the peak t i ter required 33 days and the magnitude was in fact slightly lower than a t the initial peak.

The apparent deficiency in the shark's anamnestic response called for a more incisive study. Since the secondary immunization in mammals is usually analyzed in relation to a single primary injection, i t was decided to employ a similar procedure in sharks. Much of this work is still in progress but some results are ready for reporting.

PR8 virus was injected in a single 4 ml. dose into the two lemon sharks and two adult nurse sharks and in a single 0.4 ml. dose into five baby nurse sharks delivered two days previously by Cesarean section. The results presented in FIGURE 3 show that all animals made antibody to the PR8 virus. One baby nurse and one lemon shark showed only minimal responses (1 :20 to 1 :40), but the rest found antibodies to significant t i ters of 1 :160 to 1:640. The peaks were reached by 23 days in the adult and by 30 days in the newborn nurse sharks and by 39 days in the lemon sharks. Except for the one lemon shark which had a minimal initial response, the animals failed to manifest a secondary response when the second inoculation was


made 35 to 39 days after the first. The antibody rise in the shark with the poor primary immunization may have been a reflection of a dose effect rather than a true secondary reaction.

All of the above data suggest that, based on HI antibody formation, the secondary response of sharks is either absent or deficient. A sluggish booster effect can be elicited a t five months but its nature is not clear.

The immunologic response of sharks following immunization with PR8 virus is not limited to production of HI antibodies. Although the C F reaction has so far not been obtained, neutralizing activity has been found in the sera from immunized lemon sharks. These results will be reported separately.

Immuno log ic pa t t e rns in marga tes and gars. Margates and gars were immunized with PR8 virus by several procedures and schedules. FIGURE 4 shows the points of interest with regard to immunization of margates. There appears to be a dose effect as shown by the findings that repeated injections of 2 ml. ( C ) produced higher titers than did a single injection

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of 2 ml. (A) or two injections of 0.5 ml. ( B ) . The maximum titer was attained in 10 days in most instances. In this respect the margate appears to be more efficient than the lemon and nurse shark. Yet, the maximum antibody level of the margates has been lower than that of sharks. Anti- body showed no decline for a period of a t least 40 days. As in the case of sharks there was no clear-cut evidence of secondary response when the two immunizations were spaced 30 days apart. Thus f a r no attempt has been made to follow the course of the antibody curves over extended time or to determine whether a secondary response could be elicited a t a time when antibody has declined significantly. An effort was made to increase the immunologic response of margates by incorporating PR8 virus in Freund’s complete adjuvant. The results, not given in this Figure, indicated that there was no change in the kinetics of antibody formation, but the titers reached were two to four times higher than in corresponding immunization with aqueous antigen.


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The results of immunization of gars are presented in FIGURES 5 and 6. This fresh water fish, considered phylogenetically rather primitive, produced antibodies quite efficiently. In fact, in terms of time and immunization requirements this species appeared to be more responsive than either the shark or the margate. Peak titers were obtained in 10 days or less following a single inoculation. Additional inoculations did not cause a further increase and there was no evidence of a secondary response. In terms of serum antibody concentration the maximum titers of gars were a t least as high (or higher) as those of margates, but not quite as high as the highest titers attained in lemon sharks. As in the case of other fishes the subcutaneous route of inoculation proved to be more efficacious than the intraperitoneal route, but the difference in efficacy was not as pronounced as with sharks.'

T h e n a t u r e of fish. ant ibodies . Very little is known about the chemico- physical properties of fish antibodies. Such studies have been undertaken in this laboratory and details will be published elsewhere. Meanwhile in order to round out the picture of the antiviral antibodies produced in sharks, margates and gars we are presenting a few highlights of these studies.

Sigel & Clem: Antibody Response

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M 2-Mercaptoethanol (2-ME) . The 2-ME was removed prior to antibody assay by fur ther dialysis against several changes of PBS for 18 hours a t 5°C.

With certain exceptions (which will be discussed elsewhere), this param- eter of resistance to mercaptoethanol corresponded to the sedimentation results. Thus, as shown in TABLE 1, early antibodies of the nurse shark were entirely susceptible to this treatment, whereas the later antibodies consisted of both susceptible and resistant components. The antibodies of margates and garfish were susceptible to ME both in the early and late stages of immunization. It should be emphasized tha t the ME treatment was done in the absence of iodoacetate which is known to prevent recon- struction of the globulin chains following treatment with ME.

DISCUsSION

The studies reported here show that two species of elasmobranchs, one holostean fish species, and one marine teleost fish a re capable of producing antibodies to mammalian myxoviruses. The antibody rise in the elasmobranchs, lemon sharks, was slow and reached maximum titers 30 to 40 days after initiation of immunization. These t i ters compared favorably with those usually engendered in mammals and could not be fur ther increased by continued immunization or renewed stimulation during a period of about three months. Failure to elicit a booster effect was also noted in sharks of another family, the nurse sharks. A primary-like response could however be elicited in lemon sharks upon later (a f te r five months) booster inoculation. The antibody rise in the holostean fish, the gar, and the marine teleost, margate, were much more rapid, usually reaching maximum titers in about 10 days. These t i ters were lower than those obtained with similar immunization schedules in lemon sharks. The inability to respond with enhanced antibody ti ters to secondary o r booster injections of PR8 virus given within a three-month period was also evident with these two species.

Current concepts of the nature of the secondary response view this phenomenon as an invigorated process of antibody synthesis, superseding in magnitude the process of primary immunization, generated by a greatly increased mass of antibody-forming cells. The antibody of secondary response in rabbits immunized with bacterial and soluble antigens appears to be mostly 7S, gamma. globulin.' Similarly, enduring immunologic memory to viral antigens in rabbits" and guinea pigs" depends upon the production of 7 s antibody. I n this frame of reference the fish used in this study manifested several interesting points. F i r s t and foremost they appear to have a faulty recall reaction. The margates and gars not only failed to show any evidence of anamnesis but also failed to synthesize any light antibody. The antibody produced in these species was all relatively heavy and ME sensitive and thus the failure to exhibit any signs of immunologic


memory might be explained on this basis. This assumption becomes less plausible when one considers the results of immunization of margates with BSA.7 These studies show that a vigorous secondary response could be elicited a t one month after primary stimulation with 5 mg. alum precipi- tated or 25 mg. soluble BSA. The antibody, measured by hemagglutination of tannic acid treated sheep RBC coated with BSA, rose from primary response titers of 1 :640 up to 1 :40,000. Yet this enhanced antibody synthesis was not accomplished by a shift from heavy to light antibody; rather these antibodies remained associated with the rapidly sedimenting 2-ME sensitive fractions observed with early anti-BSA and the anti-PR8 sera.

The lemon shark introduces still another deviation from what appears to be a direct relationship between 7 s antibody and immunologic memory in mammals. In this animal a delayed shift from heavy to light antibody occurs without a significant change in the kinetics or magnitude of PR8 antibody synthesis. The lemon shark response to BSA was similar to that described for PR8 in that maximum titers were obtained in about 30 days and following restimulation the shift from heavy, 2 ME sensitive to light, 2 ME resistant antibodies occurred without any evidence of increased antibody production. The anti-BSA titers obtained with the lemon shark were, however, considerably lower than in the margate.' Grey' has reported a prolonged shift from heavy (19s) to light (7s) antibody to protein antigens in the turtle without any evidence of a secondary response. Uhr, Finkel- stein, and Franklin!' reported a similar shift in goldfishes and frogs to phage @X 174 without the accompaniment of an anamnestic response. It is noteworthy that the slowly sedimenting antibody of the goldfish appeared to be sensitive to 2-Mercaptoethanol. This is a t variance with the present findings in the shark and will be subject to discussion elsewhere. Hildemann and Haas"' have, however, described a form of anamnesis in several species of fishes, i.e., homograph rejection of scale transplants. It is also interesting that the results obtained by Uhr, Finkelstein, and Frank- ling indicated that the goldfish and frog antibody was predominantly associated with the gamma2 protein. According to our data obtained so far,"' lemon sharks and gars form no antibodies which migrate as gamma2 globulin, but rather resemble gammal globulin. These animals do form gammaz- like proteins but these are devoid of antibody activity. In the margate, a species apparently lacking in proteins of gamma2 mobility, the antibodies also resemble the gammal globulins of higher animals.

The decline of heavy antibodies in margates, gars, and sharks has been noticeably slower than the decline of 19s antibody of rabbits and guinea pigs. Since the actual sedimentation coefficient of antibody from the lower vertebrates studied here has not yet been determined, the importance of this persistence is difficult to assess. I t is also not known as yet whether


this longer persistence of heavy antibodies in fish is an expression of a slower process of decay or is due to continued synthesis of new antibody.

One point tha t needs clarification, especially in relation to immunologic memory, is the antigen dose effect. In their report on studies of the pro- gression of immunologic competence in the phylogenic development of lower vertebrates, Papermaster e t al." stated tha t the horned shark (Hetero- dontiis fsanciscii ) manifested a heightened immunological response to secondary stimulation with bacteriophage and hemocyanin. This conclusion was based on accelerated clearance of antigen from the circulation and on an increased rate of phage inactivation by antisera. I t is not known at this time whether this apparent discrepancy between their results and ours is due to dosage effects, timing of the secondary stimulation, species or antigen differences, or other factors as yet undefined. The dose effect (on both anamnesis and the shift from heavy to light antibody, if the two phenomena are actually separable ) will have to be determined individually for each antigen and for each species under study.

The reasons for the differences in the immunologic responses of fishes and mammals a re unknown. It is conceivable tha t they may arise f rom several factors: the si te of the immunologic mechanism, the type of cells involved in antibody synthesis, or the nature of the proteins in which immunologic activity resides. The implications of the findings obtained to date a re sufficiently intriguing to excite fur ther investigations on the immune mechanism of poikilothermic vertebrates at the cellular and sub- cellular levels.

SUMMARY (1) Representatives of four families of two classes of fishes: the lemon

shark (Negapriori bsevimstsis) , the nurse shark (Ginglymostoma cirra- tuin ), the margate (Hawnulnn nlbtim ) , and the gar (Lepisosteus plntyrhin- czcs) were found competent to make antibodies to mammalian myxoviruses.

(2 ) The lemon sharks form HI antibodies to the human influenza PR8 virus, the equine influenza virus and the murine parainfluenza Sendai virus. The magnitude of the response to PR8 virus compared favorably with tha t of terrestrial mammals and birds. In addition, lemon sharks were also found to produce neutralizing antibodies. Studies on other species of fishes were limited to PR8 virus.

( 3 ) Maximum HI ti ters were obtained in sharks between 30 and 40 days and in gars and margates in 10 days. (4) The immune recall mechanism of these fishes appeared t o be de-

fective. In fact, during the first three months the fishes failed to manifest even a primary-like response on restimulation with PR8 antigen. After five months a renewed synthesis of antibody could be detected but was still not of the order of a secondary response.


( 5 ) The primary and renewed immune responses of these lower vertebrates differed distinctly from the responses of the rabbit and guinea pig in several other ways, including: ( a ) On prolonged immunization sharks were capable of making light, mercaptoethanol resistant antibodies in the absence of significant increases in titer. And ( b ) margates and gars continued to produce only heavy antibody despite continued immunization which in the case of PR8 virus caused no fur ther increment in t i ter , but which in the case of BSA led to definite, vigorous secondary response (studied only in the margate which still failed to make light antibody at the time of enhanced immune response following secondary stimulation).

ACKNOWLEDGMENTS It is a pleasure to acknowledge the most valuable assistance in the labora-

tory studies of Claire M. Bradshaw and Robert R. Friis , and the very effective participation in the field studies of Thomas F. McLeod, Frank A. Ferren, and Elaine C. Ferren.

REFERENCES 1. SIGEL, M. M. & L. W. CLEM. 1963. Immunological response of a n elasmo-

branch to human influenza virus. Nature 197: 315. 2. CLEM, L. W. & M. M. SIGEL. 1963. comparative immunochemical and in]-

munological reactions in marine fishes with soluble, viral and bacterial antigens. Federation Proc. 22: 1138.

3. WADDELL, G. H., M. B. TEIGLAND & M. M. SIGEL. 1963. A new influenza virus associated with equine disease. J. Am. Vet. Med Assoc. 143: 587.

4. BAUER, D. C., J. J. MATHIES & A. B. STAVITSKY. 1963. Sequences of synthesis of gamma-1 macroglobulin and gamma-2 globulin antibodies during primary and secondary responses to proteins, Salmonella antigens, and phage. J. Exptl. Med. 117: 889.

5. SVEHAG, S. E. & B. MANDEL. 1964. The formation and properties of polio virus neutralizing antibody. 11. 1 9 s and 7s antibody formation : Differ- ences in antigen dose requirement for sustained synthesis, anamnesis, and sensitivity to x-irradiation. J. Exptl. Med. 119: 21.

6. UHR, J. W. & M. S. FINKELSTEIN. 1963. Antibody formation. IV. Formation of rapidly and slowly sedimenting antibodies and immunological memory to bacteriophage gX 174. J. Exptl. Med. 117: 457.

7. CLEM, L. W. & M. M. SIGEL. 1965. Antibody responses of lower vertebrates to bovine serum albumin. Federation Proc. 24: 504.

8. GREY, H. M. 1963. Phylogeny of the immune response. Studies on some physical chemical and serologic characteristics of antibody produced in the turtle. J. Immunol. 6: 819.

9. UHR, J. W., M. S. FINKELSTEIN & E. C. FRANKLIN. 1963. Antibody response to bacteriophage gX 174 in non-mammalian vertebrates. Proc. Soc. Exptl. Biol. Med. 111: 13.

10. HILDEMANN, W. H. & R. HAAS. 1960. Comparative studies of homotrans- plantation in fishes. J. Cell. Comp. Physiol. 55: 227.

11. PAPERMASTER, B. W., R. M. CONDIE:, J. FINSTAD & R. A. GOOD. 1964. Evolu- tion of the immune response. I. The phylogenetic development of adaptive immunologic responsiveness in vertebrates. J. Exptl. Med. 119: 105.

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