CORRELATION OF C 4 AID ANTIBODY ELE9P0NSE

TO ANTIGEN-ADJUVANT INJECTIONS

APPROVED5

Majo^'frofetsor

/ * t(/t / UrZuJ^-Minor Professor

Director of the Department of Biology

Dcaii of tke Graduate Sohool

OOBEmAflOl OF 0*4 AND AHHBQDY RESPONSE

50 IHTICrEN-ADJOVAHT INJBOTIOHS

THESIS

Presented to the Graduate Council of the

forth Texan Stat® University la Partial

Fulfillment of th« Sequirtmtnts

for the Degree of

MASTia OF SOI MOB

by

Billy G. Foster, B. 3.

Denton, Texas

June, 1962

m i o f o o i f M f s

pag®

I i i s f OF I T

®tapt#r

I . IHTBOOTOIIQ* . , 1

X I . MATERIALS AID MBtHODS. 10 Start Animals Imuran! z a t l on Schedule Compliment t i t r a t i o n Agg lu t i na t i on f e s t B a c t e r i c i d a l f#-st

XIX. BB3ULIS 18

I f . DXSOUSSIOB. 32

¥„ SOHMARY . 58

BISJOSBlPSr , , . 4t

i l l

LIST OF TABLES

Tables Pag®

I. Complement Titration Procedure* 14

II. C'4 Titers In Guinea Pig Serum Following Immunization with r] Staphylococcus aureus Vaccines

III. 0*4 Titers in Guinea Pig Serum Following Immunisation with

ftttU Vaccine Following the fiegular Schedule......... 20

IV. C'4 Titers in Guinea Pig Serum following Immunization with

Sail Vaccine Following the Accelerated Schedule#.... 21

V. Oomparison of C'4 lean Values Obtained from Guinea Pig Serum Immunized with Escherichia poll and StuotelocogQui aureus Vaccines following"the Regular Injection Schedule............. 23

VI« Oomparison of 0*4 Mean Values Obtained from Guinea Pig Serum Immunized with Escherichia coll Vaccine Following the Accelerated Schedule 24

VII. Agglutination Titers in Guinea Pig Serum Following Injections with Staphylococcus aureus Vaccines 25

VIII. Agglutination Titers in Guinea Pig Serum Following Immunization with Escherichia coll Vaccine 26

IX. Bactericidal Test on Guinea Pig Serum Following Immunization with Staphylococcus aureus Vaccine as, Reported in Per Gent Kill. 28

iv

Tables Page

X. Bactericidal Test on Guinea Pig Serum following Immwnlssatlon with Ssofaerlohla qqII Vaccine as Reported in Per Oent 1111.. 29

CHAPTER I

INTRODUCTION

Some seventy years after Its discovery, complement, or

alexin, is still not completely understood# Th® discovery

of complement Is attributed to observations by Buchner (4),

who found that fresh serua often shows cytolytic propertiff.

The dual nature of this effect Is clearly recorded by lordtt

(2), who demonstrated bacteriolysis la vitro and also showed

that the bacteriolytic power of immune serum could b®

restored by addition of normal serum after It had been de-

stroyed by heating to 56 degree® centigrade, showing evidence

of two distinct substance® required for bacteriolysis* Bordet

was able to distinguish between the essential sensitl&lng

substance, or amboceptor, and a substance called complement.

The antibodies were produced or increased ®» a result of

immunization and were heat stable, while the complement was

a constituent of normal serua and was inactivated by heating.

Work of Srllch, Morgenroth (8), and Heldelberger (13) added

to this evidence that the specific antibody acts similar to

an enzyme, while the second factor, complement, acts as a

coenzyme, becoming active only after th® antigen-antibody

complex is formed#

2

la the published works of Pilleaer (19) , Brlioh and

lorganroth (8), It was asserted that the antibody, increased

by immunization, could eoabine with homologous erythrocytes

regardleiss of the presence or absence of complement to cause

hemagglutination, Eed blood cells, however, do not take up

complement unless antibody (hemolysin) Is present,and hemol-

ysis occurs only if conditions such as temperature and

concentration of reagent® are suitable.

It has been show that complement is a non-specific

substance present in all normal sera, fher# is no evidence

that more than on© kind of compliment is concerned with

hemolysis, However, there Is strong evidence that compli-

ment, which causes lysis of on® Sclnd of call (for example, the

red blood coll) is identical with that which causes lyels of

another type of cell (for Instance, the bacterium). the com-

plementary action of fresh, untreated serum from any animal

depends on the interaction of at least four separate compo-

nents* She component® are designated as 0*1, 0*2, 0'3, and

Q'4. Ivldenee is accumulating that the 0* system may be even

more complex and involve still other components. Eeeent in-

vestigation of Bruafield (3) suggest the existence of two

additional components, 0*5 and 0*6* However, these have not

been proven necessary for the hemolysis of red blood oelle *

there has been much speculation as to the production site of

thee® component®, but to date euch a site has not been proven*

3

It 1® taioim, however, that complement Is coiaposed of normel

constituent® of the circulating blood plasma.

The chemical Identity of the components of complement

b&« partially been established. 0*1 ami 0*2 were first dis-i

covered 1b 190? toy Ferrata, a® reported by Itabat and layer

(16), who separated guinea pig complement by dialysis of

serua against water into an insoluble and a soluble fraction#

0*1 and 0*2 are identified chemically as an euglobulln and

muco-euglogulin, respectively and are both heat labile, being

inaotiTated at 56 degrees centigrade in twenty minutes*

Pill«er (19) reported that when complement is treated

with cobra venom, yeast# or zymosan, an Insoluble carbohydrate

from yeast, activity is destroyed because a factor other than

0' 1 or Q'% is inactivated. fhis so-sailed third factor is

relatively heat stable* Still another thermostable factor# 0*4,

is recognised by the fact that hemolysis can no longer occur

after treatment of serum nith ammonia or primary amines.

She four ©oaponents all must be present if lysis is to

occur* fhe lytic activity of complement can be abolished by

the inactivatton of one of the components«

Since the discovery of complement, there has been con-

trasting evidence as to Aether there Is an increase or

decrease in the titer of total complement after immunization.

In the work of Pillemer (20), it nas concluded that the levels

or titer, ©f total complement remains fairly constant and i®

not raised by the immunizing process. However, In previous

4

•work of Guthrie (12), It m s reported that there is a con-

sistent increase la total complement after injections of

antigenic substance, However, this increase did not prove

to be significant. In work of Axe.lrod (1) and Dozoia (7) » it

was declared that the activity of complement Is not stimu-

lated by antigen Injections. Investigations toy Culpepper (5)

with leukemia tissue and percortin gave evidence of a general

moderate increase in both total complement and in 0*4, regard-

less of the type of tumor used. This complemented the work

of Huschel and freffers (18), who gave basis to the fact that

there was a slight rise la the total complement after injec-

tion of antigenic substances into guinea pigs. An increase

in 0*4 titer in guinea pigs after injections of oleic acid

and stearic acid was also determined in the work of Hinkle

{14) and Dowdy (6). Hilton (15) demonstrated that after in-

jections of egg albumin into guinea pigs, there waa an

Increase in 0*3 and 0*4. tenshaw (21), in turn, substan-

tiated this work of Hilton's while finding that there m s no

effect upon complement titer of guinea pigs when Injected

with small doses of cortisone, The 0*4 titer of these guinea

pigs was not increased until they had been aatigenically

stimulated. This gave an indication of the relation between

the animal's immune response and the 0*4 component of guinea

pig serum.

The antibodiei found in the sera of animals stimulated

by the introduction of an antigen into their tissue are

5

prottiaa associated primarily *lth tha grata globulin frac-

tion of the sera* Sxoapt for their ability to raact with

tpaclfio antigana, antibody globulin* usually show no

oh*»ioally raoognisabla dlffaranoa from normal globulins*

The antl gan matarlal which Initiates the production of

ballet Is uoually protein in nature, feeing of high aol«<mliir

weight; hommts antigenic a&tsrlala have bean reaogolsad *•

polysaccharides and lipids.

Antibodies a ay be sanifestad In mr&ml different ways*

They may be Jeraonstrated a® precipitin*, agglutinins, opsins,

bacteriolysina# baeterleldlae and complement fixing. 3«ith

and Oonant (23) give orelit to Grubsr and Durhaa for first

demonstrating the presence of agglutinins In ioscune serua.

Of all the serologic technlquee used in aiagaosic of infac-

tion, the agglutination teat Is the slaplaiit to perform and

haa the widest rang a of usefulness.

Certain noaantigenie substances (adjuvants), when *lxed

Tilth antigen* and Injected into anlsols, cauee a mrHed in-

crease in antibody production, The -aoet widely used adjuvant

In experimental vorlr la aalaale md occasionally la aaa is

isat®r-ln-oil eawlalon of the type 4«9crited by Freuad 111).

Yater, lanolin and antigen are mixed in mich a marker that

tha antigen Is contained in dropa of mil? surrounded by oil,

will oh effectively slows and prolong® its absorption. In

latauni action of rabbit* using guliomtllm ,iar#jl« ?reun<l,

Thoseon, Hough, scrsaar, and .?ie*ni (10) ehoved that antibody

6

production not enhanced by oleic acid and myricln but

•ma increased ?ftien added to Falba and paraffin oil# Landy

and Ullemer (17) increased protection In mice following

llpoplyaacoharlde infections, Shi* protection was correlated

•with Increased levels of perperdin. Freund (11) in hie work

•with paraffin oil as an adjuvant concluded that an increase

antibody titer m s due to the prolonged absorption of the

antigen* Sraler (9)» while working on the mechanism of Im-

munization adjuvants, denies any reaction in vitro and JL&

vivo between adjuvants and antigens* In M s opinion a de-

layed r©absorption of the antig® and-an inflamatloa at the

site of the injection are the sole activities of the adjutantst

increasing the unspeclfic resistance and enabling the organism

to react more intensely with the antigen. Preund (11) sup-

ports the conclusion that antibodies are formed at the site

of injection of antigen combined with adjuvant, and this la

In harmony with the assumption that the role of these adju-

vants is to evoke m accumulation of cells about the antigen

vhlch produces antibodies.

It m s the purpose of this paper to attempt to deter-

mine whether the Increased 0*4 previously show following

antigen and adjuvant injection could be correlated to anti-

body increase follovdag antigen injection. In this work

agglutination and bactericidal tests were used to determine

specific responses, and a correlation of these with the non-

specific 0*4 component Increase was attempted*

CHAPTER BIBLIOGHAPHY

1# Axelrod, A» B, and Prusans&y, J., "Hole of Vitamins la Antibody ?roduotion," Yitamlne and Hormones. XIII (1955), U-15.

2. Bordet, J., Resume o£ Iap|,$I> U IgSBaLSX* collected and translated by F. P. Gay, lewYork, John Wiley and Sons, Xb g«» 1901.

3. Brumfield, H. P., ttA Factor of Complement Inactivation by Histamine or Sthylenediamine,n Journal of Iaaanology. LXXII (May, 1954), 393=WT*

4. Buchner, H., "Uber die bakterlentodtened wir&ung der Zellerfrien blutserunes,* Centr. Eakit., V (1889), 817-823.

5. Culpepper, f. J., "Protective Effects of Specific lettrologou® Antimouse Tumor," unpublished master's thesis, Department of Biology, North Texas State College, Denton, Texas, 1951.

6. Dowdy, J. "Effect of Lipid Infections on Complement Titers of Guinea Pigs," unpublished master's thesis, Department of Biology, North Texas State College, Benton, Texas, 1959.

7. Bossois, T. F., "Role of Human Complement in Bacterioidal Phenomena," Journal of Immunology» XLVII (March, 1945), 229-e"^

8. Brlich, P. and Morgenroth, J., MUber haemolysins,M Berl Ciln Wooh., I, 6. Reprinted in Studies in Immunity? collected and translated by Bolduan, 0., 1-10. Sew York, John Wiley and Sons, 1910.

9. SrsXer, M., "Uber die idrJeungawtise der Adjuvant," zeitsotolft to M i f£g£&~ raental theraule (Jena). 0X711 \ 195977 294-300.

10. Freund, J*, Thomson, X. J., Hough, H. B., Soauaer, H. 7,, and ?i«anlv T. 1., "Antibody Formation and Sensi-tization with the Aid of Adjuvants," Journal of Immunology. XXX (1948), 383-398.

8

It* Frema^Jule®, "Adjuvant Action," M ^«EiT» XXYXII (1957), 18*01«

12. Guthrie, R. K«, "A Study of the Belationships Between Blood Antiprotesse System and Complement Activity la Speolfl® and ioa-speoifl© Immunological Reactions," unpublished doctoral dissertation, Departseat of Microbiology, Baylor Uaiverslty, Waco, f exas, 1954,

13. Heidelberger, M., MComplement funotioas," American Scientist. XXXIV, tl946), 597-501,

14. Hialcle, D. 0., "A Study of Aotive and Passive Iaauaity la Hornet Leukemia, unpublished master's thesis. Department of Mology, Horth teams Stat# College# Sea ton, feme, 1961*

15« BLlt«&» 13. L.» "Changes fhleh Occur la Compoaeats 0*5 and 0*4 la dulaea Pig Complement After Infections of an Antigen,M unpublished master*® thesis, Department of Mology, lorth Texas Stat® College, Denton, Texas, 1957.

Xabat, E. D., and layer* K« H., Experimental laauno-chemlstrr. Springfield, Illinois, Charles 0. fhomas,

16.

17. Laady, X., and Pillemtr, L,, "Increased Remittance to Infection and Alteration la Properdin Levels following Administration of Baoterlal Llpopoly--oohgidea, "^ourgal &£ SSgllttM, IMMlft*

18. Musehel, L. H,# and Sreffers, S* P., "Quantitative Studies on the Bactericidal Actions of Serum and Complement," &£ IVmMMM,* OH* (January, 1956), 20-27.

19. Pllliaer, Louise, Hleeent Advances in the Chemistry of Complement/ Chemical Reviews. XXXIII, Ho, 1, (August, 1 9 4 t i m c

20. Pllleaer, L# and Scker, B. 1., H An t ioomplemeatary factors In Fresh least," llfliiflhitl Chemistry. OKZTVZZ (ilaauary.

9

21* Eenshaw, !»., "Sfftet of Cortisone In3eetlons on Ooaplement fiters of Guinea Pigs,M unpublished master's thesis, Department of Biology, North fexas Stat® Qolltge* Benton, Texas, 1958.

22# Smith* 2# 5. and Conant, 1* Baoterioloar. 3rd «d«» »#w Xork, Appleton-Century« Crofta, Inc., 1952. !

Qiiksrm 11

MATERIALS AID JE8TH0D3

Guinea pigs weighing 125 to 750 grams and unselected

for sex were used aa experimental aaim&la, Sexes were sepa-

rated to prevent pregnancy which ha® been reported to cause

disturbances ia complement activity {t). Aaiaals were fed a

diet of water and Parian guinea pig chow which was supple-

mented with oats and occasionally lettuce, and were housed in

air-conditioned quarters.

for immunisation the guinea pig# were divided into

groups of four or six animals* Iwo pre-injeotion titers were

run at least three days apart to determine the titer of

agglutinating and bactericidal antibodies and 0*4 activity so

that a comparison could fee made with post injection titers,

fwo antigens were tested, staphylococcus aureus and

coll vaccines were prepared from 24 to 48 hour

old oultures grown in trypticase soy broth* After oentrifu-

gation approximately one half of the broth, was replaced with

0.9 per cent sterile -saline# Phenol was added to a final

concentration of 0.5 per cent and each vaccine m s then incu-

bated in a 37 degree centigrade water bath for 24 to 48 hours

to Insure complete kill. To this stock; vaccine sterile

10

ft

saline was added to maice an injection suspension of one to

three billion cells per milliliter#

The prepared vaccines for eaoh antigen were then divided

Into three allquots, To 20 milliliters of the diluted

vaccine in each, case was added 1.0 milliliter of the adjuvant.

The suspensions were then emulsified in the sonic oscillator

for five minutes. All vaccines were Injected by use of

sterile syringe and needle on the abdomen in 0.5 milliliter

amounts.

Immunisation Schedule

After titration of C'4 activity, agglutinating and

bactericidal antibody titers. Injections were begun, Vaooine

preparations of j|. aureus and S. ooll were used, constituting

two test series, Eaoh series consisted of groups of four to

six animals to facilitate handling. Two injection schedules

were used in both test series. One received injections

every other day for a total of three injections. This con-

stitutes the "regular schedule,* The other received

Injections every day for a total of five injections* which

will be called the "accelerated schedule,* These soheiults

were used on all groups with the exception of the animals

which received injections of j|„ aureus vaccine and paraf-

fin oil mixture. The aocelerated injection schedule was used

here.

12

Post-injection titers were run on all animals following

the regular Injection schedule one weal; after the last In-

jection. Titrations were ran five days after the last

injection on animals that were injected on the accelerated

schedule, la all eases there were at least two psst-lmmuniz-

atlon titrations ran and an. average tabulated. Each schedule

'was repeated, if necessary, to obtain successful results*

Titration of 0'4

The titration procedure was adapted from the method

presented by Kabat and layer (2). All blood specimens for

titration vera taken by cardiac punoture from unanaesthetlsed

guinea pigs# Five milliliters were taken from each animal.

The blood was then placed In the refrigerator for not less

than 30 minutes to allow the sample to clot and the serum to

separate# She samples were then centrifuge# for approximately

seven minutes to separate the serum* Serum allquota of 0,3»

0.2» and 0,15 milliliters volumes were taken from each sample*

These were treated with 0,9# 0*6, and 0,45 milliliters of

0,12 normal ammonium hydroxide solution respectively, Each

mixture was allowed to stand for 90 minutes at room temper-

ature. Ski* process inactivates 0f4 and leaves 0*1, 0*2, and

0*3 active, 'At the end of the inaetlvatlon period, 0.9* 0.6,

and 0.45 milliliters of 0,12 normal hydrochloric add respec-

tively was aided to neutralise the ammonium hydroxide. These

mixtures were then diluted with buffered saline (normal} to

13

% concentration of 1i25» 1s75 and 1i100. formal talis# was

prepared using 8.5 grams of 0, P. sodium chloride la 1,000

milliliters of deionlzed distill®# water. One milliliter of

stock buffer containing 7.5 grams of magnesium chloride aad

2,5 grams of calcium carbonate ia 100 milliliters of demln-

eralised distilled water was added. Allquots of 2.5

milliliters aad 1.9 milliliters of the Is25 dilution of la-

activated 0*4 were used ia the preparation of the 1*150 aad

Is200 volumes. Hemolysin» the anti-sheep red blood cell

serum of rabbit, preserved ia glycerine was diluted to a

1t10,000 coaceatration. Oappel sheep red blood cells were

washed with normal saliae until the superaate was clear aad

then one milliliter of the washed cells was mixed with 18

milliliters of buffered saliae. To this was added one milli-

liter of the 1s10,000 hemolysia. Shis suspension was then

inoubated in the water bath at 37 degrees centigrade for ten

minutes allowing sensitization of the cells, final dilutions

of the saline, untreated serum, inactivated 0*4 serum and

sensitized sheep red blood cells ware transferred by pipette

into test tubes as shown in the table on page 14.

tubes one, two, three, four, and five are measurements

of 0*4 activity as shown in several dilutions. 0*4 being

present ia least amount is the limiting factor, therefore

being the component measured, tube six Is a negative con-

trol and tube seven is a positive control. Tubes eight, nine,

14

mt tjrr.*© r

mooswil FOB TEST TUBS DHOTIOHS

Tube lo. Saline Treated Strum formal Serum S&BC

t 0.25 ml. 3.5 ml. of li25 0.25 ml. 1.0 ml.

n% 48m 0.25 ml. 3.5 ml. of 1*75 0.25 ml. 1.0 ml.

3 0.25 ml. 3.5 ml. of 1:100 0.25 ml. 1.0 ml.

4 0.25 ml. 3.5 ml. of 1x150 0.25 ml. 1.0 ml.

5 0,25 ml. 3.5 ml. of 1:200 0.25 ml. 1.0 ml.

6 4.0 ml. ft M # # « * 1.0 ml.

7 4.0 ml* f # # # a * 1.0 ml.

8 (%0)

8 3*0 •!# 1.0 ml. .. # * 1.0 ml.

9 3*0 ml. . 0 'ml. .. • • 1.0 *1*

10 3.0 ml. 1 • 0 . .. * * 1.0 ml.

and tea art controls for the determination of the effective*

ness of inactiv&tion of 0*4 la all three dilutions.

All tubes were then Incubated la a 37 degree centigrade

water bath for 45 mlnutee after thoroughly mixing. At tha

and of this period they were ceatrifuged to remove 'all

unlyeed cells. The supernatant of each tub® was then poured

Into a cuvette to be read In a Bauach and Lomb spectromle 20

colorimeter at 5500 A* The machine was standardized using the

negative oontrol for 100 per ©eat and the positive con-

trol to set the per cent tramsmlttanoe at sero. The per cent

transmitt&mce recordings were converted Into 50 per cent

hemolytic unite using the oonverelon table of valuee ae

15

determined by the Von trough equation found in Xabat and

Mayer (2),

Agglutination Sests

In all serum samples agglutinating antibody titers were

determined# Two pre-lmmunization titers were made in all

cases to establish the normal antibody level* In teste

following the regular schedule pott-Immunization titers were

made two wee&s after the last injection. This m i followed

three days later by a second titration* In tests following

the accelerated schedule post-immmization titrations were

run only five days after the last Injection* A second

titration m s run three days later, In some eases it was

necessary tp repeat the above Injection, schedule to secure

successful titers, In this case a two-week period l&pae was

noted before Injections were repeated. Serum for theae

titers was obtained after that for the complement titration

had been removed. Unbuffered 0*85 per cent saline was used.

Standard serial dilution agglutination technique was employed,

fhe titers were read after 25 hours Incubation in a 37 degree

oentigrade water bath and are expressed as the highest serum

dilution yielding clumping.

Bactericidal tests

Bactericidal test® were performed following the same

schedule as the agglutination test. Serum allquote were

taken from the same blood samples used In both the complement

16

and agglutination tests. Testa were m m using serum In

dilutions of 1:5, 1:10 and allquots in which 0*1 and 0*2

had been inactivated by heating to 56 degrees centigrade for

20 minutesOne tenth milliliter of the above conditions

was- added to a om tenth milliliter amount of a 24 to 48

hour old culture of J§. ooli or j3. aureus grows in trypticase

soy broth. After mi slug,the suspension was placed in a

sterile petrl dish to vhioh m i added ten milliliters of

liquid trypticase soy agar* The pour-plates vere then ro-

tated to insure even distribution of colonies, Shis method

gave counts of approximately 300 colonies. The plates then

were incubated for 48 hours at 37 degrees centigrade after

which colony counts were made on a Quebec oolony counter#

The oounts were calculated against a control which was pre**

pared by substituting one tenth milliliter of normal saline

for the serum aliquot. These counts gave an indication of

the relative capacity of the serum to cause death of the ln-

oubated organisms under the conditions defined.

CHAPTER BIBLIOGRAPHY

1. Boulanger, P., Flummer, P, J* G», Aanau, 1, B* sad Sioe, <3* I*t "Parallel Studies of Complement mi Blood Coagulation XIIi ®ie Effect of iregnaaey and Sex Hormones.* Cornell Veterinarian, XLIV (1954) 191-198• •

2, Kabat, E. D. and «&|w, 1. !#* j»«glaei*tia iBBOBt" chemistry. Springfield, Illinois, Charles 0. Thoaae, 19*i»

17

OHAPTBR III

&E3ULSB

The first group of guinea pigs were imaunlzed with

m m M l m m m s aureus vaoolaes# Bight were immunized with the

untreated vaccine, while the other seven received injections

of vaccine treated -with oleic acid. Complement titrations

before and after this Immunisation are shown la fable II# Of

the fifteen animals used In tills immunisation, all showed an

increase la 0*4 titers.

the 0 % titers of the guinea pigs la group two ar®

listed la fahle III. Sixteen animals were immunised with

Escheriohla ooll vaeolnes Injected following the regular

schedule# Five of these animals received infections of un-

treated vaccine while tlx were injected with the vaccine

previously treated with oleic acid* the remaining animals

were Iwu&ised using the m, ooll vaccine and paraffin ell#

Serum fro® ell of these anlstals was shorn to contain G'4

titer increases foilowing immunization.

Table IV shows the effect of untreated and adjuvant

treated 2*. effi.1 vaccines Injected following the accelerated

schedule# Sixteen aaiaals were used in these tests# Six

guinea pigs were tmmwxlzed, using the untreated vaccine# five

aalaals were injected with the vaccine and oleic aold, while

18

19

TABL2 II

C'4 TITBRS* II GUIHBA H 6 SBfiOH POLLOWHG IMMOTIZATIOH** mm 3!j^HIL000Q0gg

IMS VAOGT

""" falsfa! ^faoti&e vinB l *f!PL^<fPr ^ ^ fiBiliinr ^

Animals fee-Injection .

"Post-..SAliMioi

fre-In .lection

~ mat* ir.

1 10,701 16,830 12,713 16,463

2 11,029 16,823 13,015 16,665

3 11,534 17,254 13,020 16,647

4 10,010 16,483 12,673 18,213

5 10,088 15,936 13,316 14,668

6 10,758 15,736 12,679 17,169

7 10,073 15,938 11,891 16,764

8 9,863 i 16,770 Li , MI, A

••Injection* were given every day for a total of three injec-tions; "titration# were one week following the last ingestion#

the remaining tlx animal® were tested with paraffin oil and

vacoine* Bach semit showed an Increase in 0*4 activity as

compared to pre»la3e©tios titers*

a® results In all tables show 0*4 activity expressed la

50 per cent hemolytic units according to the method of Kabat

and Mayer (!)* The average pre-iamunization and po8t*imauni-

zatlon titers following each antigen serlee are given. The

frequent appearance of the value 9660 units of Q'4 wa« due to

,-t3ps value in per cent- transmission of more than 92 per cent.

Readings above this figure are not accurate.

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22

fables ? and VI show compartsons between both antigens

and adjuvants used sad their affect on the 0'4 activity, to

mean 0*4 titer of serum from animals injected with untreated

Si. aureus vaccine increased from a pre-immunlzatlon mean of

10*§09 units to a pest-iaaualaatlon mean of 16,471 unit® for

an average increase of 5,964 unit#, while the serum from

animals immunized with the vaceine treated with oleic acid

showed an increase from 12,758 to 16,656 unit®, an increase of

3,898 units.

fhe untreated ]g. coll vaccine injected on the regular

schedule showed an increase of 7«4l6 malts in guinea pig serum,

while the serum of animals injected with the vaccine and oleic

acid and paraffin oil showed an increase of 4,149 units and

81371 units, respectively. The vaccine treated and untreated

on the accelerated Injection schedule also produced an in-

crease in 0f4* &,e untreated vaccine produced an increase of

4,315 units? the paraffin oil and oleic aeld preparations an

Increase of 6,780 units and 7,841 units, respectively#

All values are averages of at least two pre- and post-

immunization titers and the time interval mad number of injec-

tions were the same.

The effect of the 3« amremr vaccines on agglutinating

antibody titers ar<a found In fable VXX. ill values are ex-

pressed as the highest serum dilution that yielded positive

clumping, ill animals are shorn to have an immune response

as indicated by their agglutination titers* All pre-iamunl-

ssation titers were negative* fhe untreated vaceine ©m both

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24

TABL3 Tl

COMPARISON OF 0*4 M M VALUES 0BUI3ED PHOM 001114 PI* m « M B

STAFHTLOOO

mmfim s c u m s

Immunization faoolae faoisime'plus' f

-Oleic Acid faooiae 'pSEStti'*41'' Paraffin Oil

Pre-Infection 9.871 10,865 1Q»5t4

Post-infection 14,186 17,645 18,365

Change •4,315 •#!?80 4.7,841

the regular and accelerated Infection schedule required three

injection series te show positive agglutination titers, file

highest titer in e&oh'ease was 11320« However, the sera:,

of animals ©a the regular Infection schedule were shorn to

have an average agglutination titer of 1:190, as compared to

11200 la the eerua of those animals Infested following tht

accelerated schedule. The vaccine plus oleic acid injected

following the regular schedule required three injection series,

while those on the accelerated schedule required only two

series. In both oases* the maximum titer m i 1i190* upon

averaging, however, those anlmais on the regular Infection

schedule showed an average of 1*175, while those following the

accelerated injection schedule had an average of 1s145. fhe

paraffin ©11 treated vaeolne was used in the acoeler&ted

schedule only and required two series of Infections to obtain

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positive agglutination* Positive test occurred in two of four

animals at a dilution of 1*1,280 with an average titer of

1»750«

Sable HII show the results of antibody titers follow**

lug injections of £• ooll vaccines# In all ease® on® infection

series % s sufficient to obtain readable agglutination titer®.

In only on# case m a there s failure to show lamuae response

to the injections, this being In the paraffin oil treated

vaccine following the accelerated injection schedule# Highest

average agglutination titers were obtained using the oleic

acid treated vaccine following the accelerated schedule, that

being tJ 160# E»e lowest average titer of it 26 was found by

using injections of the untreated vaccine on the accelerated

schedule#

fable© IX and I show the -results of the bactericidal

activity of guinea pig serum after injections of the &« aureus

and 1# coli vaccine© respectively. They are expressed in per

cent of organisms tilled as calculated against a oont-ol.

Saline had been substituted for the immune serum in this con-

trol, fhe serum from guinea pigs injected with £• ooll

following the -accelerated injection schedule showed the

highest degree of bactericidal activity, that being 50 per

cent kill. The serum showing this per cent of bactericidal

activity had been previously treated with heat to inactivate

the complement* A 1110 dilution of the same serum showed 31*3

28

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50

per cent lethal effeot, la all oasts, there was expressed a

bactericidal effect with the Inactivated complement serum*

However, with tiat eiteeptiom of one ease, there was a® lethal

effect at dilutions of 115 and 1i10 la any senna obtained by

using the j|» mrmrn raoelafs# Three crnt of the five groups

linmunlsstd with B. ooll vaeeia# showed positive results at a

dilution of it5* vhile there u s 83 far oent bactericidal

activity at'the 1s10 level.

CHAPTER BIBLIOGRAPHY

1* Kabat, E# D» and H*y«r, M. M., Experimental Immuno-ohaalgtry* Springfield, Illinois, Charla* 0. Stomas,

31

QMPTE1 If

DISCUS SI OH

Th® Inoculation of Staphylococcus aureus and Escherichia

poll vaccines Into normal healthy guinea pigs Induced a

response la the 0*4 component of complement. As noted la

fables II, III, and If, there was an Inorease In 0*4 In th®

serum of all animals after Injection of the untreated vaccines

as well as the vaccines treated with oleic aold and paraffin

oil adjuvant®. This confirms the results of Savage (4) and

Hilton (2) who have shorn that certain components of guinea

pig complement show a rise In titer following bacterial anti-

gen stimulation. The greatest Increase was found In the

serum of animal® immunized with J5» coll vaccine plus paraffin

oil adjuvant injected on the regular and accelerated schedule.

A possible explanation of this greater Increase is the presence

of the adjuvant which slow and prolongs absorption of antigen

providing a longer period of antigenic stimulation.

The nerum of most animals injected with the vaccine-adju-

vant combination either oleic or paraffin oil, showed an

increase of 0'4 greater than the Increase In the serum of

animals Injected with the untreated vaccines. This was prob-

ably due to the additional action of the adjuvant® since

injection! of adjuvants alone have been shown in the work of

32

33

Dowdy (l) to produce such an increase. The addition of the

oleic acid a® adjuvant did not produce as much Increase as

did paraffin oil, corresponding to the recognized adjuvant

action of these materials.

In agglutinating antibody tests on the serum of immunized

guinea plg®» the highest titer was found in the serum from

animals Immunized with S. aureus vaccine plus paraffin oil on

the accelerated Infection schedule. Prom this it can he seen

that the vaccine which caused the greatest increase In G'4

was not the same as that which caused the greatest Increase

in antibody activity, As noted, the highest 0*4 was in the

serum of animal® injected with 1, coll and paraffin oil. fhl®

Indicates that the 0*4 and agglutinating antibody responses

vary according to the antigen injection and are therefore

probably produced by separate mechanisms.

A comparison of the S. aureus and J§. coll vaccines with

oleic acid as adjuvant immunised on the regular schedule

showed that the mean agglutination titer was highest in the

serum of guinea pigs injected with the S. aureus vaccine, fhe

effect of this adjuvant was probably greater with the S. aureus

vaccine than the £• coll because of the lack of lipid

structural components in S. aureus which art present in J5.

fhe number of injection series each group of animals re-

ceived, that is the total amount of antigen and adjuvant, may

be of significance in determining the overall response# A

34

total of three injection series with S. aureus vaccines, pirns

oleic acid on the regular Injection schedule, was necessary to

obtain agglutination while only two series were necessary for

those animals iMaumized on the accelerated schedule. As

noted In Table ¥12, the j|. aureus vaccine and paraffin oil

combination was used only In the accelerated schedule# In

each ease, with exception of the use of the oleic acid adju-

vant, the highest titer was obtained in the serum of animals

immunized on the acoelerated schedule. Positive agglutination

titers were obtained in serua of all animals which were-in-

jected with I. coll vaccines on both injection schedule.

The results of bactericidal tests run on the serum of

guinea pigs immunised with the E. coll vaccines showed there

was a lethal effect of the immune serum in dilutions of Ii5

and 1*10 and in serum in which the complement had been inacti-

vated by heat. •' The greatest killing was found in tests where

organism® were incubated with serua from guinea pigs which had

been immunized by vacolne treated with oleic acid injected on

the accelerated schedule, fhls was true in serum diluted

1:10 and in serum In i&ich the complement had been inactivated

by heat. The effect of bactericidal activity of guinea pig

serua on 0ram negative I. coll antigen complex was to be ex-

pected, since it is known that most Gram negative organisms are

susceptible to such activity of lraune serum (5). With the

use of the vaccine and paraffin oil, there was a decrease in

bactericidal activity in the serum in which complement had

35

been inactivated as compared 'to whole seruit dilutions of 1i5

and 1*10. Such a reduotion by complement inactivation m a

expected as the usual mechanism of immune "bactericidal action

requires the presence of complement. • -

The surprising results ooourred with serum from vaccine

only injected guinea pigs and from vacolne-oleic acid Injected

animals* In these sera, the bactericidal activity w i in-

creased fey the inactivation of complement, fhis work does not

provide an explanation for suoh reversal of activity, A pos-

sible explanation could be a removal of interference with

killing hy Inactivation of only an excess of complement, while

leafing some complement activity for complement of the usual

mechanism# Or possibly the oleio acid as m adjuvant is

similar to the lipid content of the £» cell cell, and these

lipids stimulate an additional mechanism of killing not com-

pletely dependent on the presence of complete complement.

Since Oram positive organisms are not usually susceptible

to the bactericidal mechanism of immune serum {5) as -\re Gram

negative, results with j|* aureus in bactericidal tests were

surprising# In these tests whole untreated serum failed to

show any bactericidal activity when the serum was from either

vaooine-oleic acid or vaocine-paraffin oil injected animals*

Serum from £# aureus vaccine only, injected guinea figs in one

case show a 5 per oent killing action and were otherwise nega-

tive# However, when complement was inactivated by heat,

killing was noted in every serum* fhe per cent kill varied

56

fro® 9 to 17 per cent in anti-vaccine oleic acid serum to '26

par cent in anti-vaccine paraffin oil serum, to 13 to 50 par

cant in anti-vaccine only aerua. Such results have not been

previously reported, and a possible mechanism cannot be of-

fered at this time, A similar occurrence has been reported

with Slt>locoooua pneumoniae by Jeter (3). In his work he

noted killing of these Gram positive cells by immune serum

in the absence of active complement* Such results suggest a

mechani sm of immune serum as yet not explained*

In a comparison of the two vaccines tested and their

effect on 0*4 agglutination and bactericidal activity, it it

noted that the greatest increase la 8*4 and bactericidal

activity was found in serum of animals immunized with Jg* poll

vaccine while the £* aureus vaccine caused the greatest In-

crease in agglutinating antibody activity•

A comparison can also be made as to the nature of th*se

activities of the immune responses as determined by the action

of the oleic acid and paraffin oil adjuvants and the \w treated

vaccine. It is shorn that the greatest 0*4 activity was ob-

served in serum of animals Immunized Kith the vaccine-paraffin

oil complex with lower titers in untreated and anti-vaccine

oleic acid serum. The greatest bacteriddal activity was

found in serum from animals which were injected with the vac-

cine and oleic acid, This was followed closely by the

untreated vaccine and vaccine-paraffin oil combination. •

37

from these correlations, It can be stem that these

activities of the Immune response differ according to the type

of antigen complex used and that these complexes are affected

by the material used as adjuvants.

CHAPTER BIBLIOGRAPHY

1. Dowdy, J* E.» "Effect of Lipid Injections on Complement liters in Guinea rigs,* unpublished master's thesis, Department of Biology, forth Texas State College, Denton, T#xa0» 1959*

2. Hilton, D, L«, "Changes >,111 oh Occur in Components 0*3 and 0*4 in Guinea Pig Complement after Injection. of an Antigen," unpublished master's thesis, Department ©f Biology, Horth Texas State College, Denton, Texasf 1957.

3. Jeter, ¥. S., HcKee, A. P. and Mason, E# J., "Inhibition of Immune Phagocytosis of Diplooooous Pneumoniae by Human fleutrophllee nith Antibody Against Comple-ment, " firoai at a w a m . raom (»pm, 1961), 366-391 *

4« Savage, H« L., "Relation of Dosage of Antiserum to Pro-tection in Mouse Leukemia,w unpublished master*® thesis, Department of Biology, forth Texas State College, Denton, Texas, 196!.

5, Smith, 5, 8*, and Oonant, 1* Baot.eriQ.logy, 3rd ed,» lew York, Apple Crofts, Inc., 1952*

9l

38

CHAPTER V

SUMMAKT

Reunite show that there is a measurable Increase in the

0*4 titer In guinea pigs following laiauni nation by _§» aureus

$• ooli vaccines and also "with these vaccines treated with

oleic acid and paraffin oil adjuvants. fheee increase® were

demonstrated by a marked rise in Q'4 level in injected ani-

mals a® compared to ftandardlzatlon titers before injection,

fhe greatest increase in 0*4 was obtained in serum of guinea

pigs Immunized with I. eoJ4 vaccine and paraffin oil. In all

tests, greater increases were seen when adjuvants were used

as compared to untreated vaccines.

fhe results of agglutinating antibody tests show a

greater increase in activity in serum of animals injected with

H* aureus vaccine and paraffin oil* In a comparison of the

two vaccines treated with oleic acid, it ms noted that the

greater increase ws*a due to injections of the j|. aureus

vaccine, fhe effect of the adjuvant waa probably due to the

lacfe of lipid structural components in j|* Aureus which are

present in I. coll. She vaccine which caused the greatest in-

crease in 0*4 m s shown not to be the vaccine whloh caused the

greatett Increase in agglutination antibodies. This indicates

that the mechanisms involved are probably different.

39

40

Bactericidal teste showed that there was some killing

with serum of both J3, aureus and coll vaccines and with

these vaccines treated with oleic acid and paraf "in oil. The

serum of animals isreiunized with ]§* ooll-oleic acid combi-

nation showed the greatest per cent killing. ThlB w s true in

serum diluted 1:10 and in serum in which the complement had

"been inactivated with heat. A decrease in killing was found

with the use of the complement inactivated serum of animals

immunized with JI, ooli and paraffin oil as compared with the

whole aeruza, However, aureus vaccine oleic acid

serum showed an Increase in bactericidal activity when the

complement was inactivated, A possible explanation of this

could be the removal of only an excess of complement, while

leaving some complement active for bactericidal activity. Or,

possibly the oleic acid Is similar to the lipid oontent of

the jg* coll. and those lipids stimulate an additional mechan-

ism of killing*

Since Gram positive organisms are usually not suscep-

tible to bactericidal activity as Gram negatives, the resulte

of these tests were surprising. Whole untreated serum of all

test animals failed to show bactericidal activity? however,

complement Inactivated serum in all tests showed killing

ranging from 9 per cent in anti-vaoclne oleic acid serum to

50 per cent in antl-vacclne only serum. With present Infor-

mation, possible mechanisms for thete results cannot be

given*

41

la this study there was no attempt to explain the mechan-

isms Involved In these activities of the immune response tout

stuply m attempt at correlating them in respect to their

responses to antlgeit»ad;Juv&nt infections. Additional studies

will be required to determine mechanisms involved in these

events.

BIBLIOGRAPHY

BooSts

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42

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44

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