Testing the safety and effectiveness of oral administration of a live influenza vaccine*

Bull. Org. mond. Sante' 1970,42, 429-436Bull. WId Hlth Org. Testing the Safety and Effectiveness of Oral Administration of a Live Influenza Vaccine * G. I. ALEKSANDROVA, A. A. SMORODINTSEV, N. M. BELJAEVA, B. JA. VASIL'EV, R. A. GEFT, V. G. PANTELEEV, M. A. SEJFER & A. A. SELIVANOV Tests to determine the reactogenicity and immunogenicity of a live influenza vaccine when administered by mouth showed that the new method caused no reactions in adults and children and was adequately immunogenic, the results being in no way inferior to those obtained by intranasal administration. These results provide a basis for the wider use of this simpler and more convenient method ofadministration. Oral administration of the live influenza vaccine was followed by reproduction of the vaccine virus. The rapid elimination of catarrhal symptoms existing before vaccination indicates intensive production of interferon in the nasopharynx of the vaccinated persons. The standard influenza vaccine for adults induced strong reactions in children, even when the less dangerous oral route of vaccination was used. For oral vaccination ofchildren it is thus essential to use the variant of the live influenza vaccine made from further- attenuated, cold-adapted vaccine strains of influenza virus. This vaccine is quite safe and effective whether administered intranasally or by mouth. Investigations by Selivanov, Kugel' & Skrjabina (1961) showed that live polyvalent adenovirus vac- cine made from serotypes 3, 4 and 7 was highly immunogenic when given orally. The clinical reactions to vaccine that develop regularly when similar vaccine strains are admin- istered by the respiratory route were completely eliminated. These results have been confirmed by Couch et al. (1962) and Chanock et al. (1966). Since 1967, live orally-administered adenovirus vaccine and live influenza vaccine have been studied together. At first an enteric-coated preparation designed to allow the virus to pass freely through the stomach and to enter the small intestine was used (Selivanov, Gipp & Lozanovskaja, 1967). In view of the low immunogenicity of the influenza vaccine when administered by this route, a liquid influenza vac- cine, also administered orally, was subsequently used with success (Aleksandrova et al., 1968). Oral administration of a live influenza vaccine (tissue- culture type) has been studied independently at the Institute for Research on Virus Preparations, Moscow (Alekseeva et al., 1968). * From the All-Union Institute of Influenza Research of the Ministry of Health of the USSR, Leningrad, USSR. The present paper records the results of study of intranasal and oral administration of live allantoic- fluid influenza vaccine to adults and children. MATERIALS AND METHODS The vaccine Adults were vaccinated with a freeze-dried live influenza vaccine prepared at the Leningrad Research Institute for Vaccines and Sera from strains A2/21, B/95 and B/Lih which have been used in the USSR to manufacture a standard preparation for immun- izing the population over 16 years of age. The experimental batches were prepared from strains A2/133/65 and B2/67. The vaccine for oral admin- istration was used either in the dried state (for preparing an enteric-coated preparation) or in the liquid state after the dried preparation had been dissolved in a volume equal to the initial volume of allantoic fluid. Some of the experiments were carried out with the original allantoic fluid obtained from developing chick embryos infected with vaccine strains of influenza virus. Children were vaccinated with a vaccine manu- factured from variants of the vaccine strains A2/21/ 17 and B/14/17 that had been further attenuated. 2485 -429- G. I. ALEKSANDROVA AND OTHERS All these vaccines were prepared in developing chick embryos on the basis of instructions approved by the Vaccines and Sera Committee of the Ministry of Health of the USSR. Tests were made with the A2 and B monovalent vaccines or the A2+B divalent vaccine obtained by pooling equal volumes of the liquid monovalent vaccines immediately before administration. The enteric-coated vaccine was prepared by filling gelatine capsules with 0.5 g of dried vaccine. To prevent the capsules dissolving in the mouth and stomach they were processed with a 10% solution of cellulose-acetate-phthalate in acetone. Method of vaccination Two groups of healthy adults aged 20-45 years and 2 groups of children aged 3-7 years were selected by random sampling. The persons in the first group were given the live influenza vaccine and those in the second group (the controls) were given a placebo (normal allantoic fluid from developing chick embryos), which was administered in the same volume and by the same method as the vaccine. For intranasal vaccination the vaccine was diluted immediately before use in 5 times its volume of physiological salt solution and 0.25 ml of the diluted vaccine was sprayed into each nasal passage from a glass Smirnov spray. For oral vaccination a teaspoonful of the liquid preparation (2.0 ml) was given on each occasion. The vaccination was repeated 2 or 3 times at intervals of 7-10 days. For 5 days following vaccination, the vaccinated persons were kept under medical supervision. Every day they were examined and their temperatures were taken: in some cases the observations were sup- plemented by laboratory tests. Virus isolation from the throats of vaccinated persons Throat swabs were taken 24 h, 48 h, 72 h and 96 h after vaccination and the material was emulsified in 2 ml of a buffer solution. After being centrifuged for 15 min at 5000 rev/min the clarified material was injected in volumes of 0.5 ml into the allantoic cavity of chick embryos 10-12 days old. After incubation at 33°C for 48 h-72 h the allantoic fluid was subjected to the haemagglutination test with chick erythrocytes. If the result of the haemaggluti- nation test was negative a further passage of the allantoic fluid in fresh embryos was carried out. If the result of the haemagglutination test was positive, the strains isolated were identified by means of the haemagglutination-inhibition test with specific immune sera against the vaccine strains used. Isolation of the virus from the faeces of vaccinated children Faecal specimens were collected 48 h, 72 h and 96 h after vaccination and placed in flasks. The content of each flask was mixed with quartz sand, antibiotics were added (2000 IU/ml of penicillin and 1000 jig/ml of streptomycin) and the material was emulsified in 5 ml-7 ml of physiological saline and then shaken on a shaker for 15 min. The emulsion was then centrifuged and injected into the chick embryos in the same way as for isolation of virus from throat swabs. The immunogenic potency of the influenza vaccine was determined on the basis of changes in the antihaemagglutinin level in paired sera from the inoculated persons, the first taken before the first vaccination and the second 20-30 days after the final vaccination. Method of carrying out the haemagglutination- inhibition test Specimens of freshly obtained allantoic fluid from developing chick embryos infected with inhibitor- resistant strain A2/21/17 and with viruses B/14/17, B/95 and B/2/67 served as antigens in this test. The test sera were heated for 30 min at 58°C to remove any non-specific thermolabile inhibitors of haemagglutination. The heat-stable inhibitors of A2 influenza virus were not removed, since an inhibitor-resistant variant was being used in the test. Two-fold serial dilutions of the serum were made, ranging from 1: 8 to 1: 512, and placed in wells cut in Perspex panels; a dose of 4 haemagglutinating units of the working dilution of viruses A2 and B was then added in each well. The results of the test were read after 18 hours' contact at 4°C. In titrating the antibodies to the A2 virus, human red cells of the 0 group were used. Chick red cells were used in titrating antibodies to the B virus. RESULTS The reactogenicity of orally administered live in- fluenza vaccine Oral administration of liquid or enteric-coated allantoic-fluid vaccine proved fully safe for adults and caused no clinical reactions to vaccination. A total of 528 persons was vaccinated but in no case 430 SAFETY AND EFFECTIVENESS OF ORAL ADMINISTRATION OF LIVE INFLUENZA VACCINE did the temperature rise above 37.5'C. The occa- sional cases of slight temperature reactions (up to 37.5°C) were not accompanied by catarrhal symp- toms or intoxication. Clinical laboratory tests of the urine from vac- cinated persons showed no change. In the peripheral blood there was occasionally moderate neutrophilic leucocytosis and less often a tendency towards leucopenia; the white blood cell picture remained substantially unchanged. Very occasionally there was aneosinophilia, relative lymphocytosis and the occurrence of single plasmacytes. The erythrocyte counts and haemoglobin values and the erythrocyte sedimentation rates were within normal limits. The activity of alanine and aspartate transaminases in the blood serum of the vaccinated persons also re- mained within the normal range. The testing of the reactogenicity of orally ad- ministered influenza vaccine among children started in boarding schools and was continued in kinder- gartens in pre-school-age groups of children aged 3-7 years. The most detailed test was concerned with the reactogenicity for children of the further- attenuated forms of live influenza vaccine made from cold-adapted vaccine strains developed by the authors in 1962-67 and which had proved harmless for children when administered intranasally (Alek- sandrova, 1961, 1965, 1968; Smorodintsev, 1965). Table 1 shows the results of clinical examination of 256 vaccinated children aged 3-7 years and 171 children in a similarly constituted control group, drawn from the same children's establishments. Oral administration of the live vaccine to children of pre-school age proved completely harmless, even when a dose of vaccine 20 times as large as that used for intranasal administration was employed. There were only rare cases among the vaccinated children of slight reactions to vaccination in the form of a transient rise in temperature. The difference in the frequency of the more marked feverish reactions (rise of temperature to above 37.5°C) in the groups of vaccinated and unvaccinated children did not exceed 0.5 %-1.5 %. Oral vaccination of children with the further- attenuated variant of the influenza vaccine was not accompanied by any intoxication or disturbance in the general state of health. Catarrhal symptoms in the form of a cough or a cold in the nose occurred rarely and were extremely slight and short-lived. In some cases slight local inflammatory reactions devel- oped in Waldeyer's tonsillar ring and in the eyes (injection of the scleral vessels, marginal con- junctivitis, congestion of the faucial mucosa, granul- ation in the posterior wall of the pharynx, and the palatoglossal arches and sometimes swelling of the tonsils). It should be noted that these symptoms also occurred quite frequently in the unvaccinated children but their intensity was more varied. Laboratory examinations of material from the vaccinated children showed the absence of any pathological changes in the urine and only slight deviations in the peripheral blood (a moderate fluctuation in the total leucocyte count without any essential change in the white blood cell picture). The indices of transaminase activity remained at their initial level. In some cases during the post- vaccination period, a slightly positive reaction to carbon-reactive protein was discovered. All these changes in the blood were short-lived. Thus the special variant of the live influenza vaccine proved completely harmless for children aged 3-7 years. On the other hand oral administration to children of the standard live influenza vaccine used for immunizing adults led to frequent and intensive vaccination reactions in 3 %-16% of the children (Table 1). The most characteristic clinical response to oral vaccination in children was a slight and transient enlargement of the submandibular lymph nodes. This symptom occurred in 30%-70% of vaccinated children, 1 or 2 days after vaccination (Table 2). The intensity and frequency of the occurrence of this symptom were directly related to the type of influenza vaccine and were more marked with the standard vaccine. Enlargement of regional lymph nodes may be ascribed to resorption of the influenza toxin as a result of the intensive multiplication of the virus in Waldeyer's pharyngeal lymphatic ring: the virus was isolated regularly from the nasopharynx in the 4 days following the beginning of oral vaccination (Table 3). Attempts to isolate the vaccine virus from the faeces of persons to whom the liquid or enteric- coated preparation had been administered were not successful. This indicated that the virus does not reproduce actively in the intestines. Oral administration of the special variant of the live influenza vaccine to children stimulated inter- feron production. This was shown not only by the regular discovery of interferon in throat swab material but also by the interesting fact that catarrhal symptoms present in the children before vaccination were frequently eliminated. Thus in one children's 431 G. I. ALEKSANDROVA AND OTHERS E ~.°0 a (a a 0 E.° ,° \E E o Co >, -.- Z crj ._E. 0) a)Co 0C z E C,o XoE Z CL -Co 0.0 (D Z 4._ C._ CC) 0 z .o- 2 0 6 C. CD CC Co Co $-- to 0 Co 000o (Z0a ~ 6 E C0, = z L) P Co cu - 0 ZI.Co Co z Co aCo -_ - C-) 0 co .) 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L ) C-4 U04 co In 0 In I, r-w -U ') a)U CO .C- 0Q r OD o o CD 0 CO In C 0 0_ CO 0 CO C CO 0404C CO CO In In CO C-l COl CO In0:U 0) CO ) z 0 - (D z 0 -J 0 0 -J Ul 0 z I- z CD C,) w I z w IL Z 3: LJ 00 z LI 0 C) z 0 w cn z -J co -o to - o .) to E -o -o a Co CO 0 t v _ t 04 _ Lo0) co + :m 04 0 .0to IL 0 a) IL) co n: SAFETY AND EFFECTIVENESS OF ORAL ADMINISTRATION OF LIVE INFLUENZA VACCINE TABLE 2 ENLARGEMENT OF REGIONAL SUBMANDIBULAR LYMPH NODES FOLLOWING ORAL VACCINATION OF CHILDREN AGED 3-7 YEARS _ Children given vaccine by mouth Unvaccinated children No. of children in group With enlarged lymph nodes No. % 95 % ConfidenceNo. ~~~~~~limits No. of children in group ,~ ~ ~ ~ ~ ~ ~~~~~~~~~~~~~Wt enagd,yp With enlarged lymph nodes No. % Further-attenuated live influenza vaccines A2/21/17 51 22 43.1 29.35-57.75 29 0 0 B/14/17 41 12 29.3 16.13 45.54 17 0 0 A2/21117+B/14/17 111 57 51.4 41.9 -60.7 71 0 0 Standard vaccines A2/21 18 12 66.67 40.99486.66 40 0 0 B/95+ B/Li h 24 17 70.8 48.91-87.38 14 0 0 TABLE 3 ISOLATION OF THE INFLUENZA VIRUS FROM THE NASOPHARYNX OF CHILDREN VACCINATED ORALLY OR INTRANASALLY WITH MONOVALENT VACCINES A2/21/17 AND B/14/17, 48-72 HOURS AFTER VACCINATION Indices of virus isolation Method of Vaccine No. of children vaccination serotype vaccinated No. 95 % Confidencelimits Oral A2 19 13 68.4 43.45487.42 B 22 12 54.51 32.21-75.61 Intranasal A2 50 23 46.0 31.81460.68 B 50 21 42.0 28.19-56.79 establishment where vaccination coincided with increased morbidity from acute respiratory infec- tions a swift elimination of catarrhal symptoms occurred in 20 out of the 29 children vaccinated, whereas among 14 children in the control group the intensity of those symptoms increased in 6 children (Table 4). Immunogenicity of the vaccine when administered by mouth The vaccine made from further-attenuated strains of influenza virus was shown to be highly immuno- genic whether administered to children intranasally or by mouth. When children were given the mono- valent vaccine A2/21/17, or the divalent vaccine made up of strains A2/21/17 and B/14/17, by mouth an increase of 4-fold or more in the level of antibodies to the A2 vaccine variety occurred in all the sero- logically negative children. An increase in anti- bodies to the B vaccine occurred in 50% of the serologically negative children vaccinated with the monovalent vaccine B/14/17 or with the divalent A2 and B vaccine (Table 5). Table 6 summarizes the results of comparative tests in adults of the immunogenicity of the standard live influenza vaccine administered intranasally or by mouth. When the live influenza vaccine was administered as an enteric-coated preparation, and thus did not come into contact with the tissues of the upper portion of the digestive tract, the oesophagus or the stomach, it showed markedly less immuno- genic potency than when given intranasally. Oral administration of the liquid preparation, however, led to intensive production of antihaemagglutinins 7 Name of vaccine strai n 433 G. I. ALEKSANDROVA AND OTHERS TABLE 4 EFFECT OF ORAL VACCINATION WITH LIVE INFLUENZA VACCINE ON CATARRHAL SYMPTOMS IN CHILDREN Intensity of catarrhal symptoms in 5 days No. with catarrhal after vaccination Groups of children symptoms before vacci nation Disappeared Intensified No change No. % No. % No. % Vaccine given by mouth 29 20 64.0 1 3.4 8 27.6 Unvaccinated 14 2 14.3 6 42.9 6 42.9 TABLE 5 IMMUNOGENICITY OF THE FURTHER-ATTENUATED LIVE INFLUENZA VACCINES FOR CHILDREN 3-7 YEARS OF AGE WHEN THE VACCINE WAS GIVEN ORALLY OR INTRANASALLY a Serologically negative persons Virus used in Average Mean Method haemagglu- With 4-fold or more increase index of antibodyType of vaccine of tination- in antibodies increase in titre after vaccination Inhibition o antibody vaccinationtest no. N 95% Confidence levelo. % ~~limits Divalent vaccine A2+B Intranasal A2/21/17 28 25 89.3 71.77-97.73 6.5 69.0 B/14/17 154 94 61.0 53.2 -68.8 4.0 24.2 Oral A2/21/17 25 25 100.0 86.28-100.0 9.8 91.0 B/14/17 70 31 44.3 32.41-56.66 2.5 13.0 Monovalent vaccine A2 Intranasal A2/21/17 43 37 86.0 72.07-94.70 11.3 97.0 Oral A2/21/17 12 12 100.0 73.54-100.0 52.0 388.0 Monovalent vaccine B Intranasal B/114/17 30 20 66.67 47.19-82.71 7.0 42.0 Oral B/14/17 28 14 50.0 30.65-69.35 2.7 8.6 ^ Based on the haemagglutination-inhibition test with paired sera. in the blood of 53 %-68% of the persons vaccinated. These results give grounds for supposing that the tissues in the upper portion of the digestive tract are considerably more sensitive to the development of vaccine infection and the reproduction of the influ- enza virus than the intestinal tract. For that reason the oral method of administering the live influenza vaccine deserves special attention because of its great simplicity and its immunogenic effectiveness. DISCUSSION The effectiveness of the live influenza vaccine largely depends on the method of administration. The generally adopted intranasal method of ad- ministering a sufficiently high dose of attenuated virus stimulates intensive production of anti- influenza virus-neutralizing antibodies, not only in the blood, but, what is more important, in the nasopharynx of the vaccinated person, thus leading to the development of local immunity to influenzal infection in the respiratory tract. The work reported in this paper showed the effectiveness of oral administration of live influenza vaccine; this was demonstrated in adults by intensive antibody production in the blood and the complete absence of clinical reactions following vaccination. The effect was considerably weaker following vaccination with an enteric-coated preparation. This finding indicates that the vaccine virus multiplies directly in the upper portion of the digestive tract, and this is confirmed by the frequent enlargement 434 SAFETY AND EFFECTIVENESS OF ORAL ADMINISTRATION OF LIVE INFLUENZA VACCINE TABLE 6 IMMUNOGENICITY OF LIVE INFLUENZA VACCINES FOR ADULTS WHEN ADMINISTERED INTRANASALLY OR ORALLY Virus used Serologically negative persons haemagglu- Total no. With 4-fold or more increaseType of vaccine Method of vaccination tihnation- vaccinated in antibodies tinhibtion- Total no.-inhibition No 95 % Confidencetest No. ~~~~~~~~~~~~~~limits A2/133 + B/2 Oral (in capsules) A2/133 260 108 26 24.1 15.7 -32.5 B/2 265 64 6 9.4 3.52-19.30 Intranasal A2/133 204 118 52 44.1 35.1 -53.1 B/2 204 127 90 70.9 62.90-78.9 A2/21 + B/95 + BLih Oral (liquid vaccine) A2/21 190 25 17 68.0 46.50-85.05 B95 186 30 16 53.3 34.33-71.66 Intranasal A/21 150 60 27 45.0 32.12-58.39 B95 149 f 89 53 59.6 48.62-69.83 of the submandibular lymph nodes in children vaccinated with the vaccine for adults and by the ease and frequency with which the vaccine virus can be isolated from the nasopharynx of vaccinated subjects. The tests in children of the live influenza vaccine made from further-attenuated vaccine strains of A2 and B influenza established the safety and high degree of immunogenicity of that type of vaccine when given intranasally or by mouth. The standard live influenza vaccine, however, caused quite a few reactions in children even when given by mouth, although the reactions were then milder than when it was given intranasally. These results make it clear that the testing of oral live influenza vaccines in the form of a mono- valent preparation or in association with other virus vaccines should be extended to determine their effectiveness in the prevention of naturally devel- oping outbreaks of influenza. Standard egg-adapted influenza vaccine can be used for oral vaccination and as the production of this is already well estab- lished no new techniques such as the tissue cultures used by Alekseeva et al. (1968) would be required. RESUME EVALUATION DE L'INNOCUITE ET DE L'EFFICACITE DE L'ADMINISTRATION PAR VOIE BUCCALE D'UN VACCIN ANTIGRIPPAL VIVANT On a administre 'a des adultes ages de 20 'a 45 ans un vaccin antigrippal vivant du type habituellement utilise en URSS, et a des enfants de 3 a 7 ans un vaccin vivant surattenue, en utilisant soit la voie buccale soit la voie intranasale. Des groupes temoins ont requ un placebo. Donne par voie buccale, le vaccin n'a provoque aucune reaction postvaccinale chez les adultes. Chez les enfants, seuls des sympt6mes tres benins -elevation passagere de la temperature - ont et observes, meme lorsque la dose a et portee a 20 fois la dose administree par voie intranasale. On n'a constate aucune alteration notable de l'urine et du sang. Du point de vue clinique, la reaction la plus caracteristique a consiste en une hypertrophie leg&e et transitoire des ganglions lymphatiques sous-maxillaires. Ce signe, observe chez 30 a 70% des enfants, est attribue a une resorption de toxine, consequence de la multiplication intensive du virus au niveau du cercle amygdalien de Waldeyer. Le virus a ete isole regulierement du naso- pharynx pendant les 4 premiers jours suivant la vac- cination. La vaccination par voie buccale a suscite une forte production d'interferon, avec disparition frequente des sympt6mes catarrhaux presents chez certains enfants, et une reponse immunitaire aussi satisfaisante que_ la vaccination par voie intranasale. 435 436 G. I. ALEKSANDROVA AND OTHERS REFERENCES Aleksandrova, G. I. & Kugel', S. K. (1961) In: [The influenza problem. Papers read at a conference on influenza], Leningrad, p. 29 Aleksandrova, G. I. et al. (1965) Vop. Virusol., 1, 65-68 Aleksandrova, G. I. et al. (1968) In: Trudy VNII grippa MZ SSSR, vol. 2 Alekseeva, A. K. et al. (1968) In: Materialy konferencii, posvjasf6ennoj 50-letiju Sovetskoj voennoj mediciny, Vilnius, pp. 7-9 Chanock, R. M. et al. (1966) J. Amer. med. Ass., 195, 445-452 Couch, R. B. et al. (1963) Amer. Rev. resp. Dis., 88, suppl., pp. 394-403 Selivanov, A. A., Gipp, G. A. & Lozanovskaja, M. B. (1967) Trudy ItM AMN SSSR, Leningrad, 9, No. 5, pp. 84-88 Selivanov, A. A., Kugel', S. K. & Skrjabina, E. K. (1961) In: [The influenza problem. Papers read at a conference on influenza], Leningrad, pp. 30-31 Selivanov, A. A. et al. (1967) Gripp i ORZ. In: Trudy sessii institutov Virusologii imeni Ivanovskogo i VNII grippa MZ SSSR, Leningrad, pp. 202-204 Smorodintsev, A. A. et al. (1965) Industr. Med. Surg., 34, 53-64