Postgraduate Medical Journal (March 1973) 49, 185-192.

A new subunit influenza vaccine: acceptability compared withstandard vaccines and antigenicity in increasing dosage*

FREDERICK L. RUBENM.D.

GEORGE GEE JACKSONM.D.

Department of Medicine, University Hospital, Abraham Lincoln School of MedicineUniversity of Illinois College of Medicine, Chicago, Illinois

SummarySubunit influenza vaccines have the advantage ofpurity and a broad dosage range. Clinical studies weredone with a new subunit vaccine produced by detergentfractionation with tri-(n-butyl) phosphate (TNBP).Acceptability by the recipients was compared involunteers given 400 CCA units of A2 and 300 of BInfluenza vaccine as either Sharples, zonal centrifuged,ether subunit, or TNBP subunit bivalent vaccines.When given subcutaneously, subunit vaccine causedonly slightly less pain and erythema induration thanzonal or Sharples vaccine. Compared with the i.m.route, the same vaccine given subcutaneously producedtwice the local pain and eight times the erythemainduration. Fever was infrequent and low grade byeither route. HI antibody rises were good in all groups.Increasing dosages of TNBP vaccine up to 6400 CCAunits of A2 and 4800 of B given i.m. as monovalentvaccine, produced no local and few febrile responses.An augmentation of the antibody titre occurred withthe highest CCA unit dosages. Subunit vaccine giveni.m. permitted adminustration of high CCA unitdosage beyond that accepted for standard vaccinewithout any increase in local reactions and with ameasurable increase in antibody production and theinduction of antibodies against related heterologousstrains.

INFLUENZA virus vaccines have always been preparedfrom virus grown in embryonated eggs and tradition-ally consisted of inactivated whole virus. Some degreeof purification of the virus from egg contaminantswas accomplished by use ofthe Sharples internationalcentrifuge. The end-product vaccine still containeda considerable amount of egg protein and the vac-cines frequently produced local and febrile reactions.An important advance came with use of the zonalcentrifuge to further purify the vaccine virus in adensity gradient (Reimer et al., 1967). Another

* Taken from an article originally published in the Journalof Infectious Diseases (1972) 125, 656.

Requests for reprints: Frederick L. Ruben, Department ofMedicine, Montefiore Hospital, 3459 Fifth Avenue, Pitts-burgh, Pa. 15213.

advance came when the influenza virion was dis-rupted by treatment with ether. Vaccines preparedin this manner contained the envelope proteins(haemagglutinin and neuraminidase) of the -virionseparated from the nucleoproteins. Febrile responseswere diminished and the antigenicity of the virusretained (Davenport et al., 1964).

Recently a new method for disrupting the influenzavirion was reported by Neurath et al. (1970, 1971).A dispersant tri-(n-butyl) phosphate, or TNBP, wasused to release the envelope proteins of the virus.Egg constituents were removed from the viral com-ponents by column separation. Some potentialadvantages of the method over disruption of thevirus with ether are: that the TNBP-treated virusvaccine was less pyrogenic when given to rabbits,the virus was disrupted at very low v/v TNBP con-centrations, and TNBP is non-volatile and, hence,safer for mass production techniques.

In clinical trials with TNBP-produced subunitvaccine, we have compared the local and systemicreactions and the immunologic response with otherprototype influenza vaccines. With the new vaccine,high doses beyond those well-tolerated with conven-tional preparations also have been investigated.

Materials and methodsVaccinesSubunit tri-(n-butyl) phosphate vaccines were pre-

pared by Wyeth Laboratories from egg grownA2/Aichi/2/68 and B/Mass/3/66 influenza viruses.The final vaccine has essentially all of the originalviral subunits, as well as a low level (1/10,000) offormalin to inactivate any residual undisrupted virus.The standard formula (400 CCA units A2 and 300CCA B per dose) vaccines was used. Three licensedvaccines (Sharples (Wyeth'Conventional vaccine lot#1 18201), zonal ultracentrifuged (Merck-Sharp-Dohme vaccine lot $2126M), and subunit ether(Parke-Davis vaccine lot :992598 C)) were used forcomparison.

PopulationHealthy medical, nursing, and seminary students

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Frederick L. Ruben and George Gee Jackson

and new naval recruits participated on a voluntarybasis. Their ages ranged predominantly from 18 to25 years with a few older subjects. All were randomlyassigned to vaccine groups and persons with allergyto eggs or egg-products were excluded.

Immunization procedureVaccines were given by the intradermal (i.d.),

subcutaneous (s.c.) or intramuscular (i.m.) route;TNBP subunit vaccine was given by all three routes.All vaccines compared were bivalent and containedthe same amount of viral haemagglutinin. Everylocal vaccination site was inspected at 24, 48, andwhen indicated, 72 hr. The maximum intensity ofpain observed within the first 72 hr was recorded ona scale of 0-4; 0 meant no pain on palpation or

motion of the injected arm and a rating of 4 was

given for pain in the arm at rest. Oral temperatureswere taken hourly for up to 8 hr on the day ofvaccination, and at 24 and 48 hr after vaccination.Because it was not known whether adverse effectsfrom increasing dosages of subunit TNBP vaccineswould occur, the vaccines were given 'single blind',with the physician apprised of the dose given.

Safety of the subunit TNBP vaccineThe safety of the new vaccine was determined by

running biochemical studies on the volunteers' sera

for recognition of liver, renal, and haematopoietictoxicity. Sera from prevaccination, 48 hr, and 10days postvaccination were tested. The test used wasthe SMA 12/60 by Technicon Corporation, Tarry-town, New York, which included measurements ofserum transaminase (SGOT), lactic dehydrogenase,alkaline phosphatase, creatine phosphokinase, cal-cium, phosphorous, total protein, albumin, choles-terol, uric acid, creatinine and total bilirubin.

Clinical specimensWhole blood was drawn before immunization and

at an average for each group of 23-31 days post-vaccination. Nasal washings on selected volunteerswere done on the same days the sera were collected.

Haemagglunation inhibition (HI) tests

Sera were treated with receptor-destroying enzyme(RDE) (Microbiological Associates, Lot 3-3016(Titre 1: 800)) of Vibrio cholera toremove non-specificinhibitors. HI testing was done by microtechnique(Sever, 1962). Heterologous haemagglutinins wereobtained from the National Center for DiseaseControl, Atlanta, Georgia.

Neutralization tests

Sera were heat-inactivated at 56°C for 30 min,and then serially diluted in monkey kidney main-

tenance media at pH 7-6. An equal volume, 0 4 mlcontaining 100 TCID50/0O1 ml of tissue cultureadapted A./Aichi/2/68 or B/Mass/3/66 virus wasadded and the mixture allowed to sit at room tem-perature for 1 hr, then 0-2 ml of the mixture at eachserum dilution was added to each of three tubes ofmonkey kidney cells in tissue culture. The cultureswere incubated on a roller drum at 34°C for 3 days.At this time the replication of influenza virus wasdetermined by the haemadsorption test with 0-5%oguinea-pig erythrocytes.

Nasal washing specimens were lyophylized todryness, reconstituted with 3 0 ml saline, and testedfor the presence of haemoglobin by Hematest(Ames Laboratories). All positive specimens werediscarded. Aliquots of haemoglobin negative speci-mens were heat inactivated as above and handledexactly as serum for neutralization testing exceptthat 10 TCID50/01 ml was used as the virus inoculumand tubes were tested for haemadsorption after4 days. A positive nasal washing neutralization testwas defined as a four-fold rise in titre if there was aprevaccine titre, or any rise in titre if the prevaccinespecimen at 1: 2 dilution was negative. All pairednasal washings were run simultaneously.

Complement fixation testsPaired serum specimens were tested using standard

micro-titre complement fixation procedures (Sever,1962) with overnight fixation at 4°C and two fullunits of complement. A commercial soluble (S)antigen of Influenza A and B was used.

ResultsSafety of the TNBP vaccineTwo hundred and two sera representing sixty-seven

volunteers were tested for abnormalities by the SMA12/60 automated test. None of the sera tested showedabnormalities either prior to receiving the vaccine,or at 48 hr and 10 days after vaccination.

Vaccines given at conventional dosageFigure 1 shows the effect of route of administra-

tion on the production of local and systemic side-reactions from the different influenza virus vaccines.The three vaccines given s.c., Sharples, zonal and

TNBP subunit, all caused maximal pain responses ofan intensity over 2. The vaccines given i.m., thesubunit ether and subunit TNBP, had lower maximalpain responses. Erythema and induration at the localsite which averaged 4-5 cm in diameter with vaccinesgiven s.c., was hardly measurable in the i.m. vaccin-ated groups. Upper normal limits for fever wereestablished in controls as 99-40F. Fever was minimalin all groups, both for the number of subjects whohad fever and for the height of the temperature rise.

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Subunit influenza vaccine

No.Vaccine subjects

Maximum pain

Sharples 10

8o Zonal 10

Sub unit I 5TNBP _

a0 Sub unit13ether

ETSubunit

Z! TNBPI

0 2Intensity(overoge)

0 2 3 4 5

Cm diometer(tgv,eroge)

FIG. 1. Local and systemic reactions following standard dosage influenza vaccines. Allvaccines contain 400 CCA A2 and 300 CCA B.

TABLE 1. HI antibody responses to all bivalent influenza vaccines

Persons having four-fold risein HI antibodies/total Initial/final HI antibody status*

Vaccine CCA RouteA2/B A2/Aichi B/Mass A2/Aichi B/Mass

SubnitTNBP 40/30 i.d. 9/9 7/9 16/167 25/239

Sharples 400/300 s.c. 8/9 5/9 11/126 100/528Zonal 400/300 s.c. 9/10 8/10 36/214 89/852SubunitTNBP 400/300 s.c. 11/14 13/14 36/247 89/1178

SubunitEther 400/300 i. m. 11/12 11/12 85/750 89/511

SubunitTNBP 400/300 i.m. 9/10 9/10 25/513 200/1590

SubunitTNBP 800/600 i.m. 10/10 8/10 20/289 250/1820

SubunitTNBP 1600/1200 i.m. 10/10 4/5 16/390 150/2400

SubunitTNBP 3200/2400 i.m. 10/11 8/11 6/86 25/303

SubunitTNBP 6400/4800 i.m. 9/9 8/9 4/258 25/698

* Geometric mean titre (reciprocal).

Table 1 gives the frequency of the HI antibodyresponses in all vaccine groups. In the groups givenstandard dose vaccine by s.c. or i.m. routes, four-foldrises over the pre-existing titres against the homo-logous strain of Influenza A. occurred in all butseven of fifty-five subjects, and there were no signi-ficant differences among the vaccines or between thetwo routes. Six persons without initial antibody allshowed seroconversion. The number of subjects withresponses to Influenza B at conventional dosage wascomparable with that with A2 but in the combinedgroups given subunit vaccine by either route, the

seroconversion rate was greater, thirty-three ofthirty-six, than with whole virus vaccine, thirteen ofnineteen. The difference is of borderline statisticalsignificance. The geometric mean titres pre- and post-vaccine are also given in Table 1. Final titres againstA2 were higher in the i.m. groups, whereas against B,the highest titres were seen following TNBP vaccines.The average geometric fold rises in the serum titre ofHI antibody against Influenza A or B as seen inFig. 2 were similar following the different vaccineswith one exception, the increment of the response toInfluenza A2 TNBP subunit vaccine given i.m.

Temperoture (°F)No.

>99-4 Maximum

I 2 996

1000

1000

3 1008

2 1000

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188 Frederick L. Ruben and George Gee Jackson

Geometric averageVaccine Fold rise A /B

Sharples A2

Zonal B

Subunit A2

LTNBP B

Subunit A.D ether _

2 Subunitm A' t TNBP BA

0 5 1F0 15 20

Fold riseFIG. 2. HI antibody responses following standard dosageinfluenza vaccines.

which was double that observed in the other groupsand is statistically significant (P= <0 05).An additional 200 subjects received TNBP subunit

vaccine given i.m. and occasionally s.c. at standarddosage. The frequency of rises in different types orfunctions of antibodies in the serum and nasal wash-ings of randomly selected samples from these sub-jects is shown in Fig. 3. HI antibody was the mostsensitive indicator of seroconversion; responses inneutralizing antibody were nearly as frequent.Twelve of thirty sera were initially free of neutraliz-ing antibody to A2, and eleven of these showed risesfollowing vaccine. Eleven of thirty sera were free ofneutralizing antibody to B and seven of these showedrises following vaccine. Complement-fixing antibodyconversions occurred in one-fifth to one-third of

100

90

80

.......0.......

.. Neut.CF.eut

V60

t 50 3A 10

~40-A230-

20

10 'A2 BA2

0H I Neu?. CF Neut.serum serum serum nasal wash

No. lpairstested (50 30 10 9

FIG. 3. Serum and secretory antibody responses follow-ing standard dosage TNBP subunit influenza vaccine.Number vaccinated-210.

vaccinated persons tested. Only one of nine nasalwash specimens tested for neutralizing antibodyshowed a rise in titre, and this went from less than 2to a titre of 4. IgA was present in all of the specimensstudied at a concentration of 3-6-5 mg/100 ml forthe final reconstituted specimen.

Vaccines given at increasing dosageFigure 4 shows the relative freedom from side-

reactions with increasing doses of subunit vaccinegiven i.m. A small dose, 40 CCA units of A2 and 30CCA units of B in a bivalent vaccine, given intra-dermally and shown for comparison, producedstatistically significant erythema and induration,

Temperature (°F)MVaximum NolDose No. Maximum pain erythema induration >99N4 aximum

-~(CCIA A2/B) subjects>94Maiu0,E 1 40/30 9 2 99 6

400/300 0 2 100 0

800/600 0 100 4

EI 1600/1200 10 3 99-8

3200/2400 2 * 100 0

6400/4800 9 99 6

Il0 2 3 4 0 2 3 4 5

Intensity Cm diameter(overage) (overoge)

FIG. 4. Local and systemic reactions following increasing dosages of TNBP subunit influenza vaccine.

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Subunit influenza vaccine

whereas 160 times the dose given by i.m. injectionproduced virtually no local reactions or statisticallysignificant temperature elevations.As seen in Table 1, an antibody rise against A2 was

elicited in all but two of fifty persons given 400 CCAunits or more ofA2 subunit vaccine i.m. This includedseventeen persons without initial antibody toInfluenza A2 and one without antibody to B. One-tenth that dose intradermally also stimulated a risein all of nine persons. Hence, the frequency of sero-

conversion was the same with any of these doses.The geometric mean antibody titres following in-creasing dosages of TNBP vaccines showed no

correlation with the dosage either for A2 or B.Figure 5 shows the magnitude of the increment inHI antibody expressed as geometric average foldrises. All were nearly comparable except for thehighest dosage. At 6400 CCA units of A2 (with 4800CCA units of B) a striking increase in antibodyoccurred relative to the other dosages which wasstatistically significant (P= <0-05). Neutralizingantibody titres are not illustrated, but in the highestdose vaccine group they were similarly increased.None of the doses of Influenza B evoked an antibodyresponse in all of the recipients; seroconversionoccurred in only thirty-seven of forty-five personsgiven doses from 300 to 4800 CCA units. Seven ofnine responded to 30 CCA units intradermally.Higher dose elicited a greater increment in themagnitude of the antibody response, but the doseeffect was significant (P= <0-05) only at the highestdosage.

Sera from 10 days postvaccination are comparedwith the final specimens in Fig. 6. With the doses ofA2 vaccine, an abrupt break in the curve is seen

Dosage(CCA A /B)2-I

I 40/30a)

:,a

400/300

800/600

E 1600/1200E

C 3200/2400

6400/4800

Geometric averagetold rise A2/B

A2B

A2JB_A2

JB

a 2~~AB

A2B

=,-i 1B0 0 20 30 40 50 60

Fold rise

FIG. 5. HI antibody responses following increasingdosages of TNBP subunit influenza vaccine.

showing the marked increment in titre of antibodyachieved following a dose of 6400 CCA units. Asimilar but less striking effect is seen with Influenza Bvaccine at a dose of 4800. Another feature of thecurves is that with doses up to 1600 CCA units of A2,the maximum antibody response was not attaineduntil 3-4 weeks after vaccination (the final sera),whereas larger doses of vaccine produced a peaktitre earlier with an appreciable decrease after 3-4weeks.

In Table 2 are the results of tests for HI antibodyagainst heterologous strains of influenza virus. Serafrom vaccinees receiving the standard dose, 400 CCAunits of A2 and 300 CCA units of B subunit vaccinewere compared with sera from vaccinees receivingthe highest dosage, which was sixteen times greater.Testing was done with haemagglutinins from threecharacteristic isolates of A2 from earlier periods, twostrains of A1, the human prototype A (PR 8), andA (Swine); also, two earlier strains of Influenza Bwere used. The paired sera used in these tests hadshown rises to both A2/Aichi and B/Mass, the vaccine

2L)

0' >

0I

0

:c

75706560555045403530252050

5 l;

_ L/~~~~~~~~~~~~~~~~Il l l~~~~~~~~~~~~~I

0 00 0 0 0O0 00 0 0 rO

Q O00

Oo oo o~jo nrc)ooq 00 ( N\ q rn (D (\ q 0

- K) (D- t8

Influenza A2 (CCA units) Influenza B (CCA units)

FIG. 6. Early against final HI antibody responses toincreasing dosages of TNBP subunit influenza vaccine.---, 10 day specimen; , final specimen (23-31days).

TABLE 2. Heterologous influenza HI antibody responses

No. heterologous rises/no. testedVaccine subunitTNBP CCA A2/B A2* Al* Ao* B*

400/300 0/2 0/4 0/4 3/46400/4800 2/4 3/4 0/6 3/4

* Heterologous strains tested: A2; 134/64, Jap/305/57,Jap/107/62: A1; Denver/57, FM-1/47: AO; Swine/31, PR8/34:B; Great Lakes/54, Lee/40.

I

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9r0federick L. Ruben and George Gee Jacksoni

strains. Although the number of observations issmall, the lower dosage vaccine was not observed toinduce any rises in antibody that inhibited earlierheterologous A2 or A1 strains. In contrast, a four-fold or greater increase in HI antibody against thepreviously prevalent A2 and A1 strains was demon-strated in five of eight pairs of sera tested frompersons given the high dose of subunit vaccine.None of the persons had lived during the periodwhen the prototype Influenza A strains were pre-valent, and none of the sera showed the developmentof inhibiting antibody against these strains. Hetero-logous antibody responses to the earlier B strainsoccurred with Influenza B/Mass vaccine at eitherlow or high dosage.

DiscussionIn this clinical study, a subunit vaccine prepared

by a new treatment of virus harvests with a detergentTNBP and separation of the viral components on aporous column (Neurath et al., 1970, 1971) wascompared with other available vaccines. Also ofinterest was the new opportunity afforded by theacceptability of the subunit vaccine to explore theresponse over a broad range of doses up to sixteentimes the standard recommended dose.For Influenza A2 in the standard dose of 400 CCA

units, the subunit vaccine had no apparent advantageover whole virus preparations with regard to thenumber of people in whom an antibody responsewas elicited. In the case of Influenza B using 300 CCAunits, the subunit preparation was advantageous,but more extensive studies are necessary to make sucha conclusion final. Although the magnitude of therise following i.m. administration of standard doseTNBP subunit A2 vaccine was greater than thosefollowing other vaccines, the result could be fromdifferences in the average pre-vaccine antibody titre.The influence of route administration, and particu-

larly of the i.m. route, on acceptability of influenzavaccines has not received great attention in theliterature. Route has been shown to influence anti-body responses, particularly with low dosages(Tauraso et al., 1969). The subunit TNBP vaccine,when given intradermally or subcutaneously, causedreactions comparable with those produced by theSharples and zonal vaccines. These were typicalreactions of delayed hypersensitivity indicating priorsensitization of the recipients to influenza virusantigens or else local cellular recruitment with theliberation of vasoactive substances at the site ofantigen injection. Studies to be reported elsewheresuggest the former and also suggest that an increasein the number of specifically sensitized lymphocytesoccurred in the blood after vaccination. When theroute was switched to i.m., virtually no reactionswere noted with the subunit TNBP vaccine, even

including the highest CCA unit dosages given. Thisreduction in local reactions suggests that the vaccinemay have disseminated rapidly from the site ofinjection, which could result in diminished anti-genicity. No data are available to know whetherthere was a decreased stimulus to sensitization ofT-lymphocytes, but there was no decrease in anti-body response following the i.m. injection of astandard dose of subunit vaccine. In fact, the geo-metric mean response of antibody titre to the samedose of TNBP subunit Influenza A2 vaccine wasbetter by the i.m. than the s.c. route; the averagetitre rises were 25-513 against 36-247 respectively.For Influenza B also, a higher mean peak titre wasreached after i.m. than s.c. vaccine (1500 against1180), but the initial mean titre of the group also washigher (200 against 89); therefore, no difference canbe assumed.The observation that i.m. injection of subunit

vaccine eliminated the local painful reactions anddid not decrease the antibody response, permittedinvestigation of the response to increasing dosagesof vaccine. Mostow et al. (1969), in one study, com-pared high (3000 CCA units) dosages of zonal centri-fuged vaccines given subcutaneously. They found atwo- to three-fold greater incidence of adverse re-actions and a ten-fold increase in antibody titresfollowing the higher dose. Phillips et al. (1970)studied zonal centrifuged vaccines in a doserange of 200-800 CCA units and reported localreactions in 19°/ of the volunteers receiving vaccinesubcutaneously. They also showed geometric meantitres which increased with the increments in dosage.Mostow et al. (1970) in a separate study observederythema of greater than 5 cm and induration of atleast 2 cm in over 40°/ of vaccinees from 4800 CCAunits of a zonal centrifuged Influenza A2 vaccinegiven subcutaneously. A dose-response curve wasdemonstrated for HI serum antibody showing thehighest titres following maximum CCA unit dosages.Heterologous A antibody formation was also studiedand found following the higher dosages of vaccine.In our study, large doses of TNBP subunit vaccinegiven i.m. caused no intolerance, but increased theresponse only modestly, up to twenty-five fold forA2 and up to twelve-fold for B, until the highestdoses (6400 CCA units of A2 and 4800 CCA units ofB) were used.The types of antibody responses following TNBP

subunit vaccine showed that rises in neutralizingantibody paralleled those in HI antibody. Theoccurrence of some rises in complement-fixing anti-body against soluble (S) antigens of Influenza A andB is of interest. It is generally accepted that diseasealmost uniformly induced anti-(S) rises in humans.The acquisition of these antibodies followingvaccination has not frequently been tested. Since

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Subunit influenza vaccine 191

there was no clinical illness in any of the volunteersduring the study, we assume that the anti-(S) riseswere vaccine-induced, and that the vaccine containssome viral nucleoprotein. The rises confirm theobservation of Lief, Henle & Schrack (1960) thatinactivated vaccines can induce such rises. In anotherreport, data were published which showed that thestandard doses of TNBP subunit vaccine, and all ofthe other preparations, were effective in stimulatingrises in anti-neuraminidase antibody. The magnitudeof the response in general paralleled that of the HIantibody (Neurath et al., 1971). The failure of theTNBP vaccine in standard dosage to induce neutra-lizing activity in the nasal washings was disappoint-ing. Previous observations using standard vaccinehave given varied results (Phillips et al., 1970;Mostow et al., 1970; Fulk et al., 1969). Intranasaladministration of inactivated vaccine can sometimeselicit nasal antibody, but the production of localimmunity of the respiratory tract is one of thepotential advantages of live virus vaccines underdevelopment.The serum response to TNBP subunit vaccine was

rapid, especially when large CCA unit doses weregiven. These must be assumed to be secondary orbooster responses considering that most subjectshad pre-existing antibody. The few persons who hadno detectable antibody against the vaccine strain,however, also showed prompt seroconversion aftersubunit vaccine.

It is hoped that the large doses of vaccine whichproduced many fold increases in the titre of anti-bodies might also result in a persistent protectivelevel. Data in this respect are not yet available, butthe recognition of a decline in the antibody titrethree to four weeks after vaccination suggests anactive feedback mechanism that suppressed con-tinued antibody formation in the presence of hightitres in the serum. Feedback suppression seems amore likely explanation for the fall than the evolu-tion from IgM to IgG antibodies. If so, it may benecessary regardless of the dose administered toconsider combining the subunit vaccine with arespository adjuvant if persistent antibody stimula-tion and long-term protection are to be achievedwithout frequent revaccination.

Heterologous antibody responses following sub-unit vaccine administration have been clearlydemonstrated in animals (Fazekas, 1969). Suchresponses in humans following zonal centrifugedvaccines are reported in the study of Mostow et al.(1970). In our study, heterologous antibody responsesto previously encountered Influenza A and inter-related B strains were demonstrated following sub-unit TNBP vaccine. Among strains of Influenza A,heterologous antibodies were produced when a largedose of vaccine was given and were not induced by

the standard dose of 400 CCA units. It seems un-likely that small increments in CCA unitage ofInfluenza A vaccine will be useful for heterologousantibody induction. On the other hand, standarddoses of 300 CCA units of Influenza B vaccinereadily produced inhibitory antibody against theheterologous strains isolated in earlier periods.These and other biologic differences betweenInfluenza A and B viruses make it prudent to con-sider the vaccine requirements for each indepen-dently as new vaccines are formulated with thepossibility of broad variations in the range of dosage.If production of antibody against related heterolo-gous strains is a goal, a reasonable formula, accord-ing to our data, might be 300 CCA units of InfluenzaB with 6400 CCA units of A.The ability to give relatively large doses of subunit

vaccine without producing undesirable side-reactionsalso permits re-inquiry about the theoretical advan-tages of a polyvalent vaccine. If adequate doses ofantigens from past strains of Influenza A can beincluded without compromising the effective, pro-tective dose of antigens from recent strains, thelikelihood of prophylaxis against more of the naturalrecombinants of influenza viruses seems probable.A new subunit vaccine appears at least to offer thepossibility for such a study.

AcknowledgmentsThe authors acknowledge the following with appreciation

for their help with this study: Dr Robert L. Muldoon, MaryRubenis, Linda Mezny and Valentina Lolans ofthe Universityof Illinois; Captain Robert O. Peckinpaugh and CommanderRobert Mammon of the Great Lakes Naval Training Center,Great Lakes, Illinois; Dr Ben Rubin, Dr Robert Neurath,and Dr Sam Wong of Wyeth Laboratories, Incorporated.

Supported in part by U.S. Public Health Service, ResearchGrant A14059 and Training Grant A1208 and by WyethLaboratories, Inc., Philadephia, Pa.

ReferencesDAVENPORT, F.M., HENNESSY, A.V., BRANDON, F.M.,

WEBSTER, R.G., BARRETT, C.D. & LEASE, G.O. (1964).Comparison of serologic and febrile responses in humansto vaccination with Influenza A viruses on their haemag-glutinins. Journal of Laboratory and Clinical Medicine..63, 5.

FAZEKAS DE ST GROTH, S. (1969) The antigenic subunits ofinfluenza viruses. Journal of Immunology, 103, 1107.

FULK, R.V., FEDSON, D.S., HUBER, M.A., FITZPATRICK, R.,HOWAR, B.F. & KASEL, J.A. (1969) Antibody responsesin children and elderly persons following local or parenteraladministration of an inactivated influenza virus vaccine,A2 Hong Kong/68 variant. Journal of Immunology, 102,1102.

LIEF, F.S., HENLE, W. & SCHRACK, W.D. (1960) Antigenicanalysis of influenza virus by complement fixation.Journal of Immunology, 85, 483.

MOSTOW, S.R., SCHOENBAUM, S.C., DOWDLE, W.R., COLE-MAN, M.T. & KAYE, H.S. (1969) Studies with inactivatedinfluenza vaccines purified by zonal centrifugation. Bulletinof the World Health Organization, 41, 525.

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NEURATH, A.R., STASNY, J.T., RUBIN, B.A., FONTES, A.K.,PIERZCHALA, W.A., WIENER, F.P. & HARTZELL, R.W.(1970) The effect ofnonaqueous solvents on the quarternarystructure of viruses: properties of haemagglutinins ob-tained by disruption of influenza viruses with tri-(n-butyl)phosphate. Microbios, 7-8, 209.

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