Effect of dosage and route of inoculation upon antigenicity of inactivated influenza virus vaccine (Hong Kong strain) in man

Bull. Org. nmond. Sante 1969, 41, 507-516 Bull. Wid Hlth Org. | Effect of Dosage and Route of Inoculation upon Antigenicity of Inactivated Influenza Virus Vaccine (Hong Kong Strain) in Man NICOLA M. TAURASO,' RICHARD GLECKMAN,2 FRANK A. PEDREIRA,' JACOBO SABBAJ,3 RACHEL YAHWAK I & MORTON A. MADOFF 2 Earlier studies on the antibody response to inactivated influenza vaccines injected by different routes have given contradictory results, some suggesting that 0.1 ml intradermally is superior to 1.0 ml subcutaneously, others suggesting the opposite. With the advent of the 1968-69 Hong Kong influenza epidemic it seemed worth while to re-evaluate whether a smaller intradermal dose would elicit antibody responses comparable to those following a larger subcutaneous dose. A study was performed evaluating 3 doses: 0.1 ml (65 CCA), 0.25 ml (160 CCA), and 0.5 ml (320 CCA) of zonal-purified vaccine. The 0.1-ml dose was administered by both routes, and the other doses subcutaneously only. The effect of" booster " inoculation by the same route 2 and4 weeks later was also studied. Sera were examinedfor haemagglutina- tion-inhibiting antibody, and antibody response was determined by the percentage showing 4-fold or greater titre rises and by increase in geometric mean titre. The antibody response to the first inoculation was highest in the 0.1-ml intradermal groups and the lowest in the 0.1-ml subcutaneous groups. All groups receiving a second inoculation 2 weeks after the first experienced an increase in antibody response; responses to the second inoculation given 4 weeks after the first were variable. Considering the over-all effect of all combinations of doses and routes, the intradermal groups appeared to achieve the best antibody response and the 0.1-ml subcutaneous groups the least. There appeared to be an inverse relationship between antibody response andpre-immuniza- tion antibody titre. The data show that, with vaccine ofsimilar CCA content, 0.1 ml intradermally would be a reasonable alternative to, and perhaps better than, the usual 0.5-ml subcutaneous dose. The limitations of this approach are discussed. With the advent of the 1968-69 Hong Kong influenza epidemic and the subsequent shortage of vaccine, it seemed advisable to re-evaluate the sero- logical responses following subcutaneous and intra- dermal inoculation. The results of previous studies were variable. Some investigators concluded that a smaller intradermal inoculation resulted in an I Respirovirus Unit, Laboratory of Virology and Rickett- siology, Division of Biologics Standards, National Institutes of Health, Public Health Service, US Department of Health, Education, and Welfare, Bethesda, Md., USA. 2 Division of Biologic Laboratories, Laboratory Institute, Department of Public Health, Boston, Mass., USA. 3Department of Medicine, Tufts University School of Medicine, and the Medical Service, Lemuel Shattuck Hospital, Boston, Mass., USA. antibody response better than that obtained by inoculating a larger dose by the subcutaneous route (VanGelder et al., 1947; Bruyn et al., 1949b). Others found the two routes equivalent (Weller et al., 1948; Bruyn et al., 1949a; Glazer et al., 1956; Hilleman et al., 1958). Larger subcutaneous doses were found superior to smaller intradermal doses by some investigators (Woolridge & Seal ;4Boger & Liu, 1957; McCarroll & Kilbourne, 1958). Stille et al. (1959) presented evidence to show that these conflicting re- sults were due in part to variation in vaccine potency 4Woolridge, R. L. & Seal, J. R. Communication NM 005-051 06, pp. 1-12, from US Naval Medical Research Unit No. 4 (1956). 2397 -507 508 N. M. TAURASO AND OTHERS and in part to the biological difference between the intradermal and subcutaneous routes of inoculation. The antigen content of the vaccines used in some of the above studies was not known. Since then, the method of measuring the chicken cell agglutination (CCA) content of influenza vaccines has become more reproducible and our reference vaccines have been improved, so that the present vaccines are more uniformly potent than they were in the past. We do not know whether the data and conclusions of the above-mentioned studies would be valid with present vaccines. This report describes our studies on the effect of dosage and route of inoculation upon the antigenicity of inactivated Hong Kong influenza vaccine in man. MATERIALS AND METHODS Vaccine Formol-inactivated influenza virus vaccine con- taining the A2/Aichi/2/68 (Hong Kong variant) strain was kindly supplied by Eli Lilly and Company. This highly purified vaccine was prepared by con- tinuous-flow zonal ultracentrifugation. Tests per- formed in our Division of Biologics Standards laboratory showed that this vaccine contained 320 chicken cell agglutination (CCA) units per 0.5-ml dose. Volunteers Participants in this study were either (1) resident patients in a home for the aged, (2) adult workers in a laboratory, (3) adult hospital employees, or (4) university students and faculty. Admission to the study was based on a willingness to participate. Persons with a positive clinical history of allergy attributed to egg products were excluded from the study. Volunteers were fully informed of the nature of the study, and they were placed into one of 10 vaccine groups by random selection. The dose, number and route of vaccine administra- tion are shown in Table 1. All inoculations and bleedings were accomplished between 13 November and 30 December 1968. All injections were given by physicians and graduate nurses using sterile needles and syringes. Serum samples were obtained before and at 2, 4, and 6 weeks after the initial inoculation. The post-inoculation sera were obtained prior to administering the second dose when the 2 events occurred on the same occasion. Serological studies were performed only on those volunteers from whom we had a complete set of 4 sera. TABLE 1 ROUTE, DOSE AND NUMBER OF INOCULATIONS _ Interval Vaccine I nocula- D CCA No. of betweenVacroue tion oe units inocula- inocula-route per dose tions tions(weeks) I ID 0.1 65 2 2 2 ID 0.1 65 2 4 3 SC 0.1 65 2 2 4 SC 0.1 65 2 4 5 SC 0.25 160 1 - 6 SC 0.25 160 2 2 7 SC 0.25 160 2 4 8 SC 0.5 320 1 - 9 SC 0.5 320 2 2 10 SC 0.5 320 2 4 a ID= intradermal; SC= subcutaneous. The study was inaugurated at a time when there was no clinical or laboratory evidence of Hong Kong influenza disease in Massachusetts. For this reason, the presence of haemagglutination-inhibition (HI) antibodies in the volunteers is considered to have resulted from the vaccine or an exposure to the influenza antigen at some earlier time. This study was designed to compare serum antibody levels that were achieved by varying vaccine doses and routes of inoculation. No attempt was made to determine protection against infection. Serological studies The blood was allowed to clot for 1 hour at room temperature and then centrifuged at 40:C for 20 minutes at 2000 rev/min. Sera were separated and stored at -65°C until shipped in dry-ice to the Division of Biologics Standards, National Institutes of Health. Haemagglutination-inhibition (HI) tests 1 were performed using microtitre equipment (Sever, 1962) according to the following procedure: 4-8 units of antigen were added to serial 2-fold dilutions of serum which had previously been treated with receptor-des- troying-enzyme and adsorbed with cockerel red blood cells (RBC); after incubation at room temperature 1 A detailed description of methods employed in the Respirovirus Unit, Division of Biologics Standards, is avail- able on request to the senior author. INACTIVATED INFLUENZA VACCINE: EFFECT OF DOSE AND INOCULATION ROUTE TABLE 2 ANTIGENS USED IN HAEMAGGLUTINATION-INHIBITION TESTS Virus type Virus antigen A A/Swine/1976/33 A/PR/8/34 Al Al /FM/1/47 Al/Denver/l/57 Al/Ann Arbor/l/57 A2 A2/Japan/305/57 A2/Japan/1 70/62 A2/Taiwan/l/64 A2/Ann Arbor/7/67 A2/Aichi/2/68 B B/Lee/40 B/Maryland/l/59 B/Massachusetts/3/66 (23°C-25°C) for 1 hour, a 0.5% cockerel RBC suspension in 0.01 M phosphate-buffered saline was added and the mixture was incubated atroom temper- ature for 1 hour. The highest dilution of serum which completely inhibited haemagglutination was recorded as the serum antibody titre. Antigens consisted of infectious allantoic fluids obtained commercially (Table 2). Any single HI test was performed with an equal number ofcomplete serum sets from each of the vaccine groups. Computer analysis The serological results were partially analysed by computer at the Data Management Branch, Division of Computer Research and Technology, National Institutes of Health. RESULTS The number and age of volunteers are shown in Table 3. Although there were initially 323 volunteers, we were unable to obtain complete sets of 4 sera from all. The preponderance of individuals 70 years or older (62.2%) reflects the excellent co-operation given by the inhabitants of the old-age home. Antibody response to first inoculation Homologous antibody response. This is shown in Table 4 as the number and percentage of individuals with a 4-fold or greater rise in HI antibody titre to A2/Aichi/2/68. The numbers in each inoculation group were small, varyingfrom24 to 31. However, the response to the first inoculation indicated that the highest response occurred in the 0.1 ml/ID groups and TABLE 3 NUMBER AND AGE OF VOLUNTEERS Total No. in age-group (years) Vaccine No. in group gaccine 20-29 30-39 40-49 50-59 60-69 70-79 80-89 90-99 kNow 1 26 0 0 1 5 2 3 8 5 2 2 25 1 0 1 2 0 6 9 3 3 3 31 0 1 1 4 2 5 12 4 2 4 26 0 2 4 3 2 4 8 0 3 5 24 0 0 2 5 3 3 9 2 0 6 25 1 2 0 4 0 7 7 2 2 7 28 3 1 2 1 0 4 13 1 3 8 27 0 0 2 2 2 7 10 2 2 9 24 0 3 1 1 2 3 9 2 3 10 26 0 1 2 3 5 1 14 0 0 Total 1262 ] 5 [10 16 [30 18 [43 99 [21 120 509 N. M. TAURASO AND OTHERS TABLE 4 ANTIBODY RESPONSE TO HOMOLOGOUS A2/Aichi/2/68 ANTIGEN 4-fold or greater titre rise in indicated Pre- Geometric mean antibody titre of indicatedpost-inoculation serum: inoculation post-inoculation serum: a group 1st 2nd 3rd serum a 1st 2nd 3rd No. % No. % No. % GMT GMT Po/Pr GMT Po/Pr GMT Po/Pr 1 18 69 22 85 22 85 9.6 52 5.36 56 5.81 62 6.46 2 23 92 20 80 21 84 7.8 59 7.57 47 6.06 57 7.36 3 9 29 11 36 12 39 12.0 27 2.24 36 2.99 37 3.06 4 8 31 7 27 15 58 8.2 20 2.48 17 2.05 28 3.41 5 11 46 12 50 11 46 8.2 33 4.00 26 3.17 30 3.67 6 12 48 17 68 21 84 6.4 22 3.48 26 4.00 33 5.13 7 120 71 19 68 19 68 7.8 37 4.76 36 4.64 43 5.52 8 16 59 18 67 18 67 10.3 61 5.88 69 6.68 67 6.51 9 16 67 19 79 17 71 9.0 45 5.04 59 6.54 59 5.82 10 18 69 17 65 20 77 8.4 43 5.08 43 5.08 48 5.66 Total 151 57.6 t 162 61.8 176 67.2 8.7 37 4.26 38 4.39 44 5.02 a GMT = geometric mean serum antibody titre; Po/Pr = ratio of post- to pre-inoculation geometric mean serum antibody titre. the least response in the 0.1 ml/SC groups. The re- sponses in the 0.5 ml/SC and 0.25 ml/SC groups were intermediate, with the former averaging the better of the two. Similar findings were reflected in the geometric mean serum antibody titres (GMT) (Table 4). Heterologous antibody response. This response to other A2 strains was greatest to the A2/Japan/170/62 (Table 5) and less to A2/Japan/305/57 (Table 6) and A2/Taiwan/1/64 (Table 7) strains. There essentially was no response to any other antigens used, including A2/Ann Arbor/7/67 (Table 8). Antibody response to second inoculations Homologous antibody response. In those groups (5 and 8) that received a single inoculation, the anti- body response appeared to reach a peak at 4 weeks and was maintained, at least over the period of the study (Table 4). All groups (1, 3, 6, and 9) that received the second inoculation 2 weeks after the first experienced an increase in antibody response. This is reflected in the second post-inoculation sera obtained 4 weeks after the first and 2 weeks after the second inoculation. The geometric mean titres confirmed the 4-fold or greater responses. Response to the second inoculation given 4 weeks after the first was variable. Group 2 (0.1 ml/ID) and group 7 (0.25 ml/SC) did not respond, and group 4 (0.1 ml/SC) and group 10 (0.5 ml/SC) experienced an increase in antibody response. The geometric mean titres showed that all 4 groups developed more anti- body. Heterologous antibody response. This response to the A2 strains is shown in Tables 5-8. The second inoculation did not elicit heterologous antibody to those antigens not reacting after the first inoculation. Routes of inoculation Considering the antibody responses to the first inoculation and the over-all responses as reflected in the third post-inoculation sera, the intradermal route (groups 1 and 2) appeared to achieve the best results and the 0.1 ml/SC route (groups 3 and 4) was the poorest (Table 4). The other 2 dosage/route groups (i.e., 0.25 ml/SC and 0.5 ml/SC) were intermediate, with the latter appearing slightly the better. Effect ofpre-existing antibody Table 9 shows the relationship between antibody response (4-fold or greater increase in titre and geometric mean titres) and the level of pre-existing 510 511INACTIVATED INFLUENZA VACCINE: EFFECT OF DOSE AND INOCULATION ROUTE TABLE 5 ANTIBODY RESPONSE TO HETEROLOGOUS A2/Japan/170/62 ANTIGEN 4-fold or greater titre rise in indicated Geometric mean antibody titre of indicated post-inoculation serum: i re- post-inoculation serum: a Vaccineseu agroup 1st 2nd 3rd serum a 1st 2nd 3rd No. % No. % No. % GMT GMT Po/Pr GMT Po/Pr GMT Po/Pr 1 3 12 4 15 5 19 44 56 1.27 62 1.41 66 1.49 2 4 16 3 12 5 20 30 45 1.52 43 1.25 43 1.47 3 2 6.5 4 13 5 16 39 48 1.22 50 1.28 52 1.34 4 3 12 4 15 6 23 36 47 1.31 42 1.17 53 1.49 5 2 8 2 8 5 21 33 42 1.26 39 1.19 43 1.30 6 3 12 3 12 3 12 38 45 1.18 41 1.09 49 1.28 7 6 21 9 32 8 29 49 69 1.41 76 1.56 78 1.60 8 5 19 6 22 8 30 33 52 1.59 59 1.80 67 2.05 9 3 13 4 17 4 17 30 38 1.26 44 1.46 51 1.68 10 4 15 5 19 5 19 27 47 1.70 44 1.62 47 1.70 Total 35 13.3 44 16.8 54 20.6 36 48 1.36 49 1.37 54 1.53 titre. a GMT = geometric mean serum antibody titre; Po/Pr = ratio of post- to pre-inoculation geometric mean serum antibody TABLE 6 ANTIBODY RESPONSE TO HETEROLOGOUS A2/Japan/305/57 ANTIGEN 4-fold or greater titre rise in indicated post-inoculation serum: I1st 2nd 3rd No. % No. % No. % 2 7.7 4 16 2 6.5 1 3.8 2 8.3 2 8.0 4 14 7 26 4 17 7 27 35 13.3 4 15 3 12 5 16 2 7.7 3 13 3 12 6 21 5 19 3 13 3 12 37 14.1 4 15 6 24 5 16 4 15 4 17 3 12 2 7.1 5 19 4 17 5 19 42 16.0 Pre- inoculation serum a GMT 50 35 49 33 39 34 64 36 37 33 40 Geometric mean antibody titre of indicated post-inoculation serum: a 1st GMT Po/Pr 62 1.24 51 1.47 56 1.14 39 1.17 43 1.09 39 1.15 76 1.19 64 1.80 49 1.33 53 1.62 2nd 3rd GMT Po/Pr GMT Po/Pr 55 1.08 41 1.18 60 1.22 37 1.11 45 1.16 43 1.25 80 1.25 56 1.59 43 1.16 48 1.45 53 1.30 50 1.24 66 1.31 56 1.60 56 1.14 44 1.34 42 1.06 51 1.52 62 0.98 55 1.55 57 1.54 45 1.38 53 1.32 a GMT = geometric mean serum antibody titre; Po/Pr = ratio of post- to pre-inoculation geometric mean serum antibody titre. Vaccine group 2 3 4 5 6 7 8 9 10 Total 512 N. M. TAURASO AND OTHERS TABLE 7 ANTIBODY RESPONSE TO HETEROLOGOUS A2/Taiwan/1/64 ANTIGEN 4-fold or greater titre rise in indicated Pre- Geometric mean antibody titre of indicated post-inoculation serum: inoculation post-inoculation serum: a Vaccineseuagroup 1st 2nd 3rd serum a 1st 2nd 3rd No. % No. % No. % GMT GMT Po/Pr GMT Po/Pr GMT Po/Pr 1 4 15 3 12 4 15 23 32 1.41 30 1.31 32 1.41 2 3 12 2 8.0 3 12 24 31 1.32 30 1.25 30 1.25 3 3 9.7 7 23 6 19 23 29 1.25 36 1.53 34 1.43 4 1 3.8 3 12 6 23 21 22 1.03 27 1.31 32 1.53 5 1 4.2 0 0 1 4.2 21 26 1.26 23 1.12 24 1.16 6 2 8.0 3 12 3 12 25 30 1.21 34 1.36 30 1.21 7 5 18 6 21 7 25 33 42 1.28 42 1.28 48 1.45 8 2 7.4 7 26 4 15 30 34 1.14 50 1.67 42 1.43 9 3 13 3 13 4 17 21 31 1.50 31 1.50 34 1.63 10 2 7.7 2 7.7 2 7.7 22 30 1.41 27 1.24 29 1.34 Total 26 9.9 36 13.7 40 15.3 24 30 1.27 32 1.35 33 1.38 a GMT = geometric mean serum antibody titre; Po/Pr = ratio of post- to pre-inoculation geometric mean serum antibody titre. TABLE 8 ANTIBODY RESPONSE TO HETEROLOGOUS A2/ANN ARBOR/7/67 ANTIGEN 4-fold or greater titre rise in indicated Pre- Geometric mean antibody titre of indicatedpost-inoculation serum: inoculation post-inoculation serum: a Vaccine -______ -serum a group 1st 2nd 3rd 1st 2nd 3rd No. % No. % No. % GMT GMT Po/Pr GMT Po/Pr GMT Po/Pr 1 1 3.8 2 7.7 1 3.8 9.1 11 1.17 11 1.24 10 1.11 2 0 0 0 0 0 0 8.7 10 1.15 9.2 1.06 8.7 1.00 3 0 0 1 3.2 0 0 9.6 10 1.05 9.8 1.02 10 1.05 4 0 0 0 0 0 0 8.4 8.2 0.97 8.0 0.95 8.2 0.97 5 0 0 0 0 1 4.2 9.0 9.0 1.00 8.5 0.94 9.0 1.00 6 0 0 0 0 1 4.0 8.7 8.7 1.00 8.7 1.00 9.5 1.09 7 1 3.6 1 3.6 0 0 9.1 11 1.16 9.8 1.08 9.3 1.03 8 1 3.7 1 3.7 1 3.7 10 11 1.08 12 1.14 12 1.11 9 1 4.2 1 4.2 2 8.3 8.5 10 1.22 9.5 1.12 9.5 1.12 10 2 7.7 1 3.8 1 3.8 8.4 11 1.32 10 1.21 9.6 1.14 Total 6 2.3 7 2.7 f 7 2.7 9.0 10 1.11 9.6 1.07 9.5 1.06 a GMT = geometric mean serum antibody titre; Po/Pr - ratio of post- to pre-inoculation geometric mean serum antibody titre. INACTIVATED INFLUENZA VACCINE: EFFECT OF DOSE AND INOCULATION ROUTE TABLE 9 ANTIBODY RESPONSE RELATED TO PRE-IMMUNIZATION ANTIBODY TITRE 4-fold or greater titre rise Geometric mean antibody titre of indicated Per- in IndicatedpoticuaonsrmPre- Total centage post-inoculation serum: post-inoculation serum: immuniza- -ases_of_totaltion titre caeIf oa St 2nd 3rd 1st 2nd 3rd Post Po/Pr Post Po/Pr Post Po/Pr 4 131 50 No. 84 91 100 22 5.4 23 5.8 29 7.2 % 64 70 76 8 52 20 No. 40 40 42 47 5.9 44 5.4 47 5.9 % 77 77 81 16 29 11 No. 13 15 17 47 2.9 54 3.4 56 3.5 % 45 52 59 32 24 9.2 No. 8 6 8 72 2.2 64 2.0 66 2.1 64 20 7.6 No. 6 8 7 124 1.9 128 2.0 133 2.1 % 30 40 35 128 4 1.5 No. 0 2 2 256 2.0 304 2.4 304 2.4 % 0 50 50 256 1 0.4 No. 0 0 0 512 2.0 512 2.0 256 1.0 % 0 0 0 512 1 0.4 No. 0 0 0 512 1.0 512 1.0 512 1.0 % 0 0 0 Total 262 No. 151 162 176 37 4.26 38 4.39 44 5.02 [62 67 a Post = geometric mean serum antibody titre of post-inoculation sera; Po/Pr = ratio of post- to pre-inoculation geometric mean serum antibody titre. homologous antibody. There appears to be an inverse relationship between antibody response and pre-immunization antibody titre. In addition Table 9 discloses that a large number of volunteers had pre- existing antibody to the A2/Aichi/2/68 antigen, reflecting the fact that elderly people probably had prior contact with a closely related strain. Statistical analysis The data in Table 4 on the homologous antibody responses of post-inoculation sera obtained at 2 and 6 weeks were statistically analysed (Table 10). For the purposes of this analysis individuals were considered as members of 4 dose/route groups: statistical group I consisted of vaccine groups 1 and 2 (0.1 ml/ID); group II of vaccine groups 3 and 4 (0.1 ml/SC); group III of vaccine groups 5, 6 and 7 (0.25 ml/SC); and group IV of vaccine groups 8, 9 and 10 (0.5 ml/SC). Considering the 4-fold or greater antibody titre rise responses to the first inoculation, as determined from serological tests performed on the first post-inoculation sera, the difference between groups I and II was highly significant at the 1 % level. Comparing the 3 SC groups (II, III, and IV), the differences were also significant at the 1% level. Although the difference between groups II and III was significant, the difference between groups III and IV was not. Considering the over-all responses of these same groups to the combination of doses, number and routes of inoculation, as determined from sero- logical tests performed on the third post-inoculation sera, only the difference between groups I and II was significant at the 1 % level (Table 10). Relationship of age to antibody response Although it had not been initially intended, the study population was skewed toward the older age- group. This allowed us to evaluate the immunization procedures in individuals for whom influenza vaccine is medically indicated. However, because people aged 65 years and older had evidence of previous exposure to antigens related to the Hong Kong variant, it became very important to determine 13 513 514 N. M. TAURASO AND OTHERS TABLE 10 RESULTS OF STATISTICAL ANALYSIS OF 4-FOLD OR GREATER HOMOLOGOUS ANTIBODY RESPONSES Post-inoculation serum Statistical Vaccine 1st a 3rd bgroups group Dose and route compared No.|Significant No D.F. Significant 2 D F atlI% level atlI% level I and I and 2; 0.1 ml/ID Highly 27.61 1 Yes 12.62 1 II 3 and 4 0.1 ml/SC I I, 3 and 4; 0.1 ml/SC III and 5, 6 and 7; 0.25 ml/SC Yes 17.03 4 No 8.66 4 IV 8,9 and 10 0.5 ml/SC 11 and 3 and 4; 0.1 ml/SC Yes 8.75 1 No 4.83 1 III 5, 6 and 7 0.25 ml/SC III and 5, 6 and 7; 0.25 ml/SC No 1.32 1 No 0.5 1 IV 8, 9 and 10 0.5 ml/SC I and 1 and 2; 0.1 ml/ID No 3.44 1 No 1.27 1 IV 8, 9 and 10 0.5 ml/SC a Analysis of data from Table 4, column 3, reflects the responses to the first vaccine inoculation. b Analysis of data from Table 4, column 7, reflects the responses to the combination of doses and routes. how this affected the results. Since the presence of sidered. The majority of volunteers (66 %) belonged antibody to the A2/Aichi/2/68 antigen was rare in to the older age-group (Table 11). This group also volunteers aged 64 years or less, 2 broad age- had a geometric mean titre which was 2.5 times that groups-namely, 20-64 and >65 years-were con- of the younger age-group (Table 12). Considering TABLE 11 NUMBER AND PERCENTAGE OF INDIVIDUALS WITH 4-FOLD OR GREATER RISE IN ANTIBODY TITRE TO A2/Aichl/2/68 ANTIGEN WITH RESPECT TO AGE Age-group 4-fold or greater titre rise in indicated post-inoculation serum Vaccinie 1St 2nd 3rd grouo 20-64 years >65 years 20-64 years >65 years 20-64 years >65 years 20-64 years >65 years No. % No. % No. % No. % No. % No. % No. % No. % 1 8 31 16 62 4 50 12 75 6 75 14 88 6 75 14 88 2 4 16 18 72 3 75 17 94 2 50 15 83 4 100 15 83 3 8 26 21 68 3 38 6 29 4 50 7 33 5 63 6 29 4 9 35 14 54 2 22 4 29 2 22 4 29 4 44 9 64 5 7 29 17 71 3 43 8 47 2 29 10 59 1 14 10 59 6 7 28 16 64 2 29 9 56 4 57 11 69 6 86 13 81 7 7 25 18 64 4 43 14 78 4 43 13 72 3 43 13 72 8 5 19 20 74 3 60 11 55 4 80 17 85 3 60 13 65 9 6 25 15 63 5 83 9 60 6 100 10 67 5 83 10 67 10 9 35 17 65 5 56 13 77 7 78 10 59 9 100 11 65 Total 70 27 172 66 34 49 103 60 41 59 111 65 46 66 114 66 INACTIVATED INFLUENZA VACCINE: EFFECT OF DOSE AND INOCULATION ROUTE TABLE 12 GEOMETRIC MEAN SERUM ANTIBODY TITRES TO A2/Aichi/2/68 ANTIGEN WITH RESPECT TO AGE Geometric Geometric mean titre or ratio of indicated post-inoculation serum mean titre of Titre Ratio Vaccine pre-inoculation 1st 2nd 3r_|1t__2d |_r group a serum 1St 2nd 3rd I1St 2nd 3rd 20-64 >65 20-64 >65 20-64 >65 20-64 >65 20-64 >65 20-64 >65 20-64 >65 years years years years years years years years years years years years years years 1 4.4 14 14 87 16 95 19 103 3.1 6.4 3.7 7.0 4.4 7.7 2 4.0 10 16 94 14 69 45 75 4.0 9.3 3.4 6.9 11 7.4 3 4.8 19 11 43 19 54 21 53 2.4 2.3 4.0 2.9 4.4 2.8 4 4.0 15 8.6 35 8.6 26 13 43 2.2 2.3 2.2 1.7 3.1 2.8 5 4.0 11 11 52 9.8 39 7.3 54 2.7 4.7 2.4 3.5 1.8 4.9 6 4.0 8.4 11 31 12 35 20 43 2.7 3.7 3.0 4.2 4.9 5.2 7 5.9 9.7 16 47 16 47 20 51 2.7 4.9 2.7 4.9 3.3 5.2 8 5.3 14 18 87 18 108 37 85 3.5 6.5 3.5 108 7.0 6.3 9 7.1 11.1 57 39 72 51 57 51 8.0 3.5 10 4.6 8.0 4.6 10 4.7 12 22 61 24 59 44 50 4.7 5.3 5.0 5.1 9.3 4.3 Total 4.7 12 15 55 17 55 22 59 3.2 4.5 3.6 4.6 4.8 4.9 a See Table 11 for the numbers and percentages of those aged 20-64 years and >65 years within each vaccine group. the fact that individuals with no detectable A2/Aichi/ 2/68 antibody were recorded as having a titre of equal to or less than 1: 4, this difference between the 2 age-groups could actually be much higher. The 4-fold or greater rise in antibody and the geometric mean titre ratio data shown in the totals rows of Tables 11 and 12 respectively tend to sup- port the conclusion that individuals 65 years or older developed antibody quicker than the younger age-groups, as determined from tests performed on sera obtained 2 and 4 weeks after the initial vaccine inoculation. However, by the sixth week after inoculation the only difference between the 2 age-groups was a higher geometric mean titre in the older individuals (Table 12). Since the geometric mean titre fold- increases were similar, the higher geometric mean titre in those 65 years or older probably reflects the higher antibody titres found in their pre-inoculation sera. DISCUSSION When influenza virus undergoes a major antigenic shift, all efforts are made to manufacture and dis- tribute sufficient vaccine to protect man from the oncoming epidemic. The interval between the ap- pearance of a new strain in the community and the occurrence of major epidemics is determined by nature. This interval is usually short and, if a sub- sequent shortage of vaccine occurs, emotional reac- tion is high. It would be helpful if it were possible to make each vaccine lot go farther. It has been known that small intradermal injec- tions will often result in an immune response equi- valent to larger subcutaneous doses (Martin, 1942). Studies to evaluate the relative effectiveness of both routes for the administration of inactivated influenza vaccine have resulted in variable results. VanGelder et al. (1947) reported that a single O.1-ml intradermal dose of inactivated influenza vaccine resulted in a level of serum antibody several times that obtained by a single 1.O-ml dose of the same vaccine admin- istered subcutaneously. The antigen content of the vaccine used was not reported. Bruyn et al. (1949b) also concluded that intradermal inoculation resulted in an antibody response better than that obtained following the subcutaneous route. Although Rend- torff et al. (1959) did not evaluate the subcutaneous, 515 516 N. M. TAURASO AND OTHERS route, they reported that intradermal inoculation was effective in preventing influenza. Using vaccines con- taining 500 CCA units/ml, Woolridge & Seal (op. cit.) and Boger & Liu (1957) found the antibody res- ponse to subcutaneous inoculation superior to that by the intradermal route. McCarroll & Kilbourne (1958), using an Asian influenza virus vaccine containing 200 CCA units/ml, concluded that intra- dermal injection of 0.1-ml and 0.2-ml doses offered no advantage over similar doses administered sub- cutaneously. A problem with this study was that the antibody response to the 200 CCA unit vaccine was considered suboptimal. Weller et al. (1948), Bruyn et al. (1949a), Glazer et al. (1956), and Hille- man et al. (1958), using vaccines of different antigen content, essentially found that smaller intradermal doses were equivalent to larger subcutaneous doses in eliciting an antibody response. In our experiments zonal-purified vaccine con- taining 320 CCA units per the usual 0.5-ml sub- cutaneous dose was used. Our results suggest that intradermal inoculation of vaccine in an amount equivalent to one-fifth the subcutaneous dose should be considered. This could easily be accomplished when the usual subcutaneous dose is contained in 0.5 ml, as it is in the newer purified vaccines. A disturbing feature about the use of the intra- dermal route is that it is technically complicated and requires well-trained personnel to perform the injections. If one were to administer a 0.1-ml dose by the intradermal route but inadvertently inject it subcutaneously the recipient, instead of receiving his immunization by the better route, would receive it by the less favourable one. There is little doubt from our data that in man the response to subcutaneous injection of inactivated influenza virus vaccine is dose-dependent-that is, the response increases with higher doses. Regarding the interval between doses when more than 1 dose is contemplated, it appears that a booster antibody response does occur when the second dose is given 2weeks after the first. When a community awaits an epidemic, it would be ideal to give the second dose as soon as it would boost the antibody response. ACKNOWLEDGEMENTS The authors are indebted to Dr Barbara Ganem of the Lemuel Shattuck Hospital, Jamaica Plain, Mass.; Dr Curtis Prout of the Harvard University Health Services, Cambridge, Mass.; Dr Arthur Linenthal of the Hebrew Rehabilitation Center for Aged, Jamaica Plain, Mass. and Dr J. Feeney of the Harvard Medical School Health Services, Boston, Mass., and the laboratory technicians of their respective medical facilities. We thank Mrs Rosma Venerbrandts and her staff of the Biostatistics Depart- ment of the Lemuel Shattuck Hospital for their help in randomizing the study. We also thank Miss M. Darlene Myer and Mr Dennis A. George of the Data Management Branch, Division of Computer Research and Technology, National Institutes of Health, for their assistance in obtaining the data in good time. We are particularly grateful to Mrs Ann Bennett, who assisted in the design of the study, and to Roy Trimmer, Eldridge Staton and William Barthlow for their technical assistance in per- forming the antibody assays. REFERENCES Boger, W. P. & Liu, 0. C. (1957) J. Amer. med. Ass., 196, 1687-1689 Bruyn, H. B., Meiklejohn, G. & Brainerd, H. D. (1949a) Amer. J. Dis. Child., 77, 149-163 Bruyn H. B., Meiklejohn, G. & Brainerd, H. D. (1949b) J. Immunol., 62, 1-10 Glazer, M. W., Benenson, A. S. & Wheeler, R. E. (1956) Pediatrics, 17, 482-487 Hilleman, M. R., Flatley, F. J., Anderson, S. A. & Luecking, M. L. (1958) J. 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