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Amplification of phage neutralization by complement, antiglobulin, and antiallotype sera
VII~OI,OGY
46, 797-807
(1971j
Amplification
of AntiGlobulin,
l?. L. ADLER,
Phage
Neutralization and
by
AntiAllotype
W. S. WALKER,’
Complement, Sera’
.\NI) RI. FISHMAN
Rabbits and rats injected once with lo9 PFU of coliphage T2 (0.1 ,~g nitrogen) I’+ spend with neutralizing antibody which during the first 2 weeks is minimally efficient. Complement, antiglobulin, or antiallotype antibody amplify the activity of such early IgM and IgG antibodies up to 1000.fold. and the kinetics of the early antibod? response measured in the presence of these enhancing reagents closely resemble each other but differ from the kinetics of the neutralizing antibody response determined irr t,he conventional manner. 1IVTROI)UCTIOIV
t*he course of our studies on t,he induction of antibody formation against T2 bacteriophage in vitro, the need for highly sensitive assays attuned to the detection of “early” antibodies arose. A passive hemagglutination assay n-as developed which filled this requirement8in part (Walker et al., 1969, 1971), and a neutralization test that employs complement or antiglobulin as amplifying agents has been explored. A role for complement’ or some of its component)sin the nrutralization of viruses by nnt,ibody has received sporadic at,tention in the litcruturc wer since J. H. Mueller’s observation (1931) on the role of “alexin” in the neut,ralization of vaccinia virus. Ginsberg and Horsfall (1949) found that’ calcium ion and thcrmolabilc serum components contributed to the neutralization of myxoviruses by normal sera. It was shown subsequently that dwomplcmc~ntation removed the IWUlrnlizing activity of normal swa for the T In
’ This work was supported in part by (+rant AI-06890 from the Xational Institute of Allergy and Infert,ious Iliseases, and by Grant GH-29018X from the National Srience Foundation. * Supported by Training Grant AI-00408 from the National Institute of Allergy and Infectious Diseases. 797
phages of Escherichia coli (Cowtrl, 1962), and t’hat both complement and specific* antibody were required (Toussaint and Muschcl, 1962; Muschel and Toussaint, 1962). Antibody appearing early in immunization, like normal antibody, was shown to depend on complement, not only in t I](. neutralizat’ion of T phagcs (~lus~l~~~land Toussnint, 1962; HBjek, 1969), but also in the inactivation of myxoviruscs (St,yk, 1962; Smordintsw and ‘L’abrov, 1963; Lirwot t and Lrvinson, 1969), hrrpcs simpltlx virus (Yoshino and Taniguchi, 196n; Hcineman, 1967; Hnmpar et al., 1968) and arboviruws (Ozaki :md Tabrbyi, 1967; Way and Gnrnw, 1970). The neutralizing activity of Ig1I as w11 as that of “early” IgG ant,ibody is c~nhanwtl by wmplcment (Yoshino and Tanigwhi, 1965; Hampar et al., 196X; H&j&, 1969), and rewnt worlc has shown that only t h(k first’ wmponents of complement are required for amplification (Linscot t and LcvinscIII, 1969; Daniels ebal., 1970). Another series of rcccnt papers prwwfs data on the neutralization of infectiw complexes of virus-ant’ibody by antiglobulin reagclnts (Hampar et al., 1968). The data to be prescntc>dconfirm and cxxtclnd thrw findings.
798
ADLER,
WALKER,
AND
FISHMAN
The antiglobulin used in most experiments was prepared by intensive immunization of Immune sera. Concentrates of coliphage rats with a fraction of rabbit serum globulin T2 (2 X 1012 PFU/ml) were prepared as rich in IgM (first peak from Sephadex Gpreviously described (Walker et al., 1969), 150). Precipitin analysis in agar gel showed and rabbits and rats were injected intravethis serum to react both with purified IgG nously or intraperitoneally with lo9 PFU and IgM fractions. After absorption with an (0.1 pg nitrogen) in 1.0 ml. Selected immune excess of IgG it still precipitated IgM, sugsera were decomplemented by incubation gesting that it contained antibodies of anti-k with preformed immune complexes of bovine specificity as well as antibodies to light serum albumin (BSA) and rabbit anti-BSA polypeptide chains shared by IgG and IgJ1. formed at equivalence. Amplification by antiallotype serum. The Complement. Pooled sera from normal rats procedure was exactly the same as that used selected for low “background” anti-T2 neufor antiglobulin. Reagents used in the prestralizing antibodies were used in fmal diluent investigation were prepared by intensive tions of 1: 20 or 1:40. For experiments immunization of appropriate rabbits wit’h requiring higher concentrations of complerabbit r-globulin. The anti-4 reagent was ment, sera were obtained from rats subjected made in al,al/b5,b5 rabbits by injection of to whole body X-irradiation (650 R) and al,al/b4,b4 globulin, the anti-6 reagent by bled 5 days later. Individual sera from such globulin into al,a3/ animals were screened for their lack of sig- injecting al,u3/b6,b6 b5,b5 rabbits and the anti-5 serum by immunificant T2 neutralizing activity in a final nization of al,al/b4,b4 rabbits with al,al/ dilution of 1: 10, as well as for their ability b&b5 globulin. The anti-l (053P) and anti-3 to amplify an early anti-T2 complement(E-275-6) sera were kindly donated by Dr. dependent immune serum. Appropriate sera were pooled and stored in small volumes at Rose Mage. All were potent sera and were used after heat-inactivation (56” for 30 min) -70”. Rabbit sera, obtained 5 days after and lo-fold dilution. whole-body X-irradiation of 1000 R, were found to be as effective as rat sera in enhancRESULTS ing complement-dependent neutralizing activity. In all instances, the dose of compleComplement Dependence of Early Antibody ment used in the assay of antibody was after a Single Injection of Phage chosen so that no more than 10 % of the test A group of 12 rabbits each received a dose of phage was neutralized by complesingle intravenous injection of lo9 PFU of ment alone. T2. Serial bleedings were titrated for their AmpliJication by complement. Serially di- neutralizing activity with or without the luted antisera or tissue culture fluids, in addition of complement, with the resulbs 0.4~ml amounts, were incubated for 30 min shown in Fig. 1. It will be noted that 50% at 37” after addition of 0.2 ml of suitably neutralization titers NTE~ measured in the diluted complement and 0.2 ml of a phage absence of added complement rose, at a suspension diluted to contain 400 PFU of T2. moderate rate, to a mean of 160 on day 11 Duplicate or triplicate aliquots of 0.2 ml and then showed no further significant were then plated on E. coli B. Further de- increase to day 35. In contrast, titrations in tails are described in the text. the presence of added complement showed a AmplQication by antiglobulin. Serial dilurapid rise in activity which attained a peak tions of antiserum or tissue culture fluid of 10,240-20,480 on day 5 or 6, maintained (0.4 ml) were incubated with 0.2 ml of phage itself at that level until about day 15-20 suspension (400 PFU) for 30 min at 37” and then declined. The most effective amfollowed by a second incubation period of plification by complement occurred in the 90 min at 4”. A potent antiglobulin serum bleedings obtained between days 4 and 15 (heated 56” for 30 min) was then added (0.4 when titers were 128-256 fold greater in ml of 1: lo), and after an incubation for 30 the presence than in the absence of added min at 37” aliquots were plated as above. complement (0.2 ml 1: 10). Thereafter the MATERIALS
AND
METHODS
AMPLIFIED
NEUTRAI,IZ,1TTON
TR!)
amplification was less; on day 35 it merely averaged 4-fold. Hyperimmune sera (Walker et al., 1969) showed a similarly small amplification of their neutralizing act,ivities. The da$a just preser&ed suggested that neutralizing activity of the earliest antibody, like that of the antibody found aft(er day 15, was not as effect’ively amplified by complement as that of the antibody found in the intervening period. The data presented in Fig. 2 show that the amount of complement used in the previous experiments raised t,he NT&o of a Z-day bleeding from 32 t,o 128, 5 mere $-fold rise. By increasing the complement dose 16-fold amplification was obtained. Decomplementation of the serum, on the other hand, led to a, 4-fold decrease of NT50, Since the change in regard to complement amplification appeared to be within the IgM class of antibodies, as will be discussed in the next section, it appeared of interest to determine whether “late” IgM antibody resembles t’hat found on day 2 or that found during the period of maximally enhanceable IgM antibody. A purified IgM fraction from a al-day bleeding was examined, and it was found that its neut,ralizing activity was ampli~ed 2~0-fold by complement. It appears, therefore, that IgM antibody formed between day 4 and approximately day 15 is enhanceable by complement to a significantly greater degree than IgM formed before or after this period. Changes in specificity, affinity, or in complement fixing ability may be responsible singly or in combination, and furt.her work is needed to resolve this problem.
at OD 250 nm. A pool of fractions 3-7 was designated IgM, and fractions 11.-U ant1 14-17 were pooled to yield IgG prepnra tions I and II. To confirm the ahsencc of IgM ant,ibody in the t,wo IgG prepar~~tiol~s, :I pool of fractions 11-17 was subject,ed to a second centrifugation in a sucrose gradient. Fractions l&l5 from this gradient ~ontainetl neutralization activity (7 S) I\-hicall was readily enhanced by complement. As shown by the data in Table 1, both IgM and IgG antibody from this serum was an~plified in its neutralizing activity, the IgM antibody more effectively than IgG. These data, t,ogether wit’h those shown in Figs. 1 and 2, are in agreement with the results of others (H&jek, 1969) and show Ohnt while IgM antibody present on day 11 (days 4-15) is most readily amplified, the IgG ant*ibody found on day I1 is enhanced no less than the TgLM antibody found prior to day 4 and not si~ificantly less t,han IgM in the 2X-day bleeding.
ImmunogEobulin
COMPLEWENT AMPMFICXTION OF NEUTU \LIZATIOK IJY IgM AND IgG ANTIRODTIW --.- -. ~~-..
Class of Neutralizing
Anti-
body Enhanced by Complement
While little or no neutralizing IgG antibody could be found in sera obtained during the first week after immunization, the llday bleeding permitted a comparative study of IgM and IgG neutralizing antibodies with respect to complement amplification. The serum was placed on a lO-37% linear sucrose gradient and was subjected to 16-hr centrifugation at 32,000 rpm in a SW 50.1 Spinco rotor. The contents of each gradient tube were collected in 20 equal fractions. 6 and 17, respectively, had absorption peaks
Titration of Complementfor Amplifying tivity
.-1c-
If the technique of complement-ampl.ified neutralization is to yield meaningful results as a measure of antibod~T activity, it is is important to ascertain that antibody and not t)he accessory factors determine the end point,. The presence of varying amounts of normal antibody reactive with T2 phage in the sera of nonimmunized rats imposed an upper limit on the amount of complement that could be used without complicating TABLE
50% Globulin classa
IgM IgG IgG
pool pool pool (L Sucrose
I IL
1
-
Neutralization _-i
jcomppment
/ C;;$-
fractions
.-._
;im$l?p-
!-.--_-.-_---_~__-^! / 1632 i 2048-4~~ 4-x / 128 I j 4-8 128 gradient
titer .-- .._ .I
128X 16-32X 16--32X . ..--.._-II-day bleed-
from
ing. 6 Complement, source: dilution 1:40 (25 ~.rl).
Normal
rat
scrltm,
final
800
ADLER,
WALKER,
AND
FISHMAN
. .
.
.
A
Y
l A
m l
0 l
FIG. 2. T2 neutralizing activity of a 4.day bleeding from a rabbit that received 109 PFU of T2. The serum was assay untreated (a), after decomplementation (0), decomplemented followed by the addition of rat complement, final dilution 1:40 (X) or rat complement from X-irradiated animals, final dilution 1:lO (A).
complement of extremely low “normal” antibody content, and it was possible to use such serum as complement in amounts of up to 200 /.ll. Figure 3 shows the composite results of complement titrations in which serial dilutions of a decomplemented rat antiserum to T2, obtained 4 days after the intraperitoneal injection of log PFU, were titrated in the the assay. By the use of pools of sera from presence of varying amounts of rat compleselected rats, this upper limit was 20-40 ~1 ment. The larger dosesof complement (25in the standard test as described in the 200 fil) were from a pool of sera from Xprevious figures and table. Several attempts irridiat,ed rats. It may be seenthat maximal were made to remove the “normal” phage- amplification (512-1024-fold) was att,ained neutralizing antibody from sera that were wiyith 100 ul of complement and that a to be used as complemenL Absorptions measurable degree of amplification required with phage that had been inactivated by only 1.25 ~1. It will also be noted that the irradiation or by treatment w&h formalin experiments already discussed were done in were carried out in the cold to preserve the presence of that amount of complement complement activit’y. It was found that (20 .ul) which represents the upper limit of such treatment frequently resulted in en- the linear portion of the plot shown in Fig. 3. hanced “neutralizing” activity which proved to be independent of complement and, on The Inhibitory E$ect of Phosphate Ions further examination, could be shown t)o be In titrating the sera from normal rats it a result of the introduction into the serum was found that t,hose with NTso of less of solubilized E. coli antigens. The use of than 4 when diluted in phosphate buffer sera from rats that had been subjected to (2.25 mM) often neutralized T2 phage tot’al body X-irradiation (650 R) provided when they were diluted 44Sfold in buffer FIG. 1. T2 neutralizing
activity of rabbit sera following a single intravenous injection of lo9 PFU T2 on day 0. Each symbol stands for the titer of 1 rabbit serum obtained on the day indicated. Assayed in the absence (0) or presence (A) of 0.02 ml of active normal rat serum as complement source.
AMPLIFIED
SO1
NEUTIIALIZATIC)PU’ TABLE
PhosphateY (m&0 _.---_-
2
Percent neutralization bv ran anti-T2 antibody ifinal dilution ib __--~-.__ -..-~~ ~-.-... 1:250 1:12.50 1: 6250 _____ -----.--. ~_-.~ ..-.__ - -
99 98 30 96 R-2 !N 100 !).i ::i !I.-> 3X 9') IN 96 3I 97 87 2.i 90 51 7 I!) II 80 _--~..__ ---.Q Phosphate buffer, pH i.3. * Each reaction mixtllre (0.8 ml) contained 0.4 ml of diluted rat anti-T2 serum, 0.2 ml of rat complement 1: 10, and 0.2 ml phase suspension. The Mg2+ and Ca2+ were 0.25 m&’ and 0.075 m.W. wspectively. 0 0.75 1.5 3.0 6.0 12.0 24.0 37.5
3. Neutralizing activity of a decomplemented rat antiserum obtained 4 days after int,raperitoneal injection of 109 PFU of T2. Assayed in the presence of increasing amounts of rat complement. FIG.
lacking phosphate and containing magnesium and calcium (0.5 and 0.15 &I’, respect,ively). To investigat’e the apparent dependence of complement-enhanced neut’ralizing activity on suitable Ca2+ and lMg2+ concentraGons, and to ascertain the antagonistic effect of PO&, serum obtained from rats 4 days after a single injection of lo9 PFU of phage was titrated with complement,, Mg2;2f and CM?+ at, constant concent’rations while POa2- and antibody were varied. The results, summarized in Table 2, showed that under the condit,ions chosen PO?- concentrations of 12 n& or higher inhibited si~ificantl~ and that this effect was most pronounced when limiting amounts of antibody were approached. The NT60 of the serum, in the absence of added complemerit, was 40-80. It is clear, therefore, that the inhibitory effect of POF, although marked, was not absolute within the conwntration range tested.
The enhanced rwuralization of rort~ain viruses by antibody in the presence 11f complement has been attributed to the effect of the added bulk of the ahsorbtrd protein on absorption or penetration of the virus in its reaet,ion -tvith cells (Daniels ct ui., 1970; Linscott and Levinson, 1969j. Wc considered alternate or additional mwhanisms, such as possible effects of cornpI+ ment on the rate of conformational changc~s in antibody that occur secondarily to its reaction v&h the virus (Jshizaka and Campbell, 1959; Laffert-y, 1963; Svehag and Blotlr, 1967 and others), or on the ~socia~i{)~~ rate of virus and antibody. I’re~irnirI~~r~ experiments yielded results which illustrated certain difficulties in applying convent ion aI kinetic analysis procedures to L4t~arly” mtibodies. Summarized in Table 3 are data obt:rined with a 5 day bleeding from a rabbit3 immuniz~ with T2. To allow comparisor~~ of the degree of Ileut,ra~izatio~l of phage plat cd dire&Iv with results obt,ained by plating aft>er dilution, the range of phage conwntrations was restricted to S-fold. It will be noted that the neutralization reaction closely followed the “percentage law” (Andrcws and Elford, 1933) except -\l-hen plating wts
802
ADLER,
WALKER,
AND
TABLE REACTIVATION
FISHMAN
3
OF NEUTRALIZED
VIRUS
BY DILUTIOLY
Phage input Anti-T2 serum=
1:20 1:40 1:80
976
2% Undil.”
II%.
le (1OO)f 13 (95) 76 (70)
8 (99) 33 (93) 109 (77)
1952
Undil.
Di1.d
Undil.
Dil.
13 (99) 60 (94) 223 (77)
32 (96) 176 (82) 520 (47)
29 (99) 140 (93) TMTCQ
269 (85) 664 (66) 1040 (47)
a Rabbit serum (5 day) following log PFU T2 IV. Values given are final dilutions. b Total number of phage added to incubation mixtures. c Plated without dilution. d Diluted prior to plating. e Total PFU in volume plates. f Values in parentheses are percent neutralization of phage input. 0 Too many to count. TABLE EFFECT
OF COMPLEMENT OF NEUTRALIZED
4
ON VIRUS
THE REACTIVATION BY DILUTION
I
Total PFU T2 added 840 1 8,404 1 84,000 1840,000
~-
1:20 1:40 1:80 1:4000 1:8000
+ + + + +
82b (34)” 100 100 74 (28) 100 100 99 37 (12) 100 99 100 98 93 (62) 89 91 (30)
(47)
(34)
(& (84) 04) (82) (50)
(& (82) (20) (83) (23)
(12) (19)
0 Rabbit serum obtained 5 days after intravenous injection of 109 PFU T2. Values shown are final dilutions. b Percent neutralization in sample plated without dilution. c Values in parentheses are percent neutralization in sample diluted lo-fold or more before plating.
preceded by dilution. If the dilution step was interposed, marked reactivation took place unless the antibody concentration was very high (antiserum 1: 20). To test the effect of complement upon dissociation by dilution, a number of experiments was performed which yielded data of which those in Table 4 are representative. It is apparent from these data that dilution prior to plating (lo-fold or more) brought about marked reactivation of “neutralized
virus” from reaction mixtures, even from those which contained high levels of antibody but no complement. The data further show that the reactivation by dilution affected a fixed percentage of “neutralized virus” over a 1006fold range of virus concentrations. In the presence of complement and high antibody concentration, there was no dissociation upon dilution. When antibody approached limiting concentrations, complement no longer prevented dissociation upon dilution. Complement, therefore, appears to reinforce the dissociable bond between virus and antibody. Comparative Studies on Amplification by Complementand Antiglobulin Since antiglobulin has been successfully employed in amplifying the neutralizing activity of antisera against certain viruses (Hampar et al., 19SS), leading to the suggestion that this reagent acts like complement by adding bulk to the virus-antibody complex, some experiments were done to compare these reagents. An added reason was provided by the unfortunate presence of high levels of anticomplementary activity in tissue culture fluids from cultures in which antibody against T2 phage had been induced in vitro by the method previously described (Adler et al., 1966). This activity, often still present in fluids diluted 8-16-fold, appeared to be concentrated in particulate material which could be spun down in 90
AMPLIFIED
NEUTRALIZATION TABLE
-~
AMPLIFKXTION
OF RABBIT
ANTI-%!
50% Bleedinga
5
NEUTKALIZISG
Neutralization
SO:<
ACTIVITT
BY AXTIIUBBIT
Amplification
titer
(day) No antiglobulin 2
Antiglobulin
40 160 160 160-320 160-320 40,000-80,000
6
15 21 35 Hype@
320-640 40,960 20,48040,960 20,480-40,960 40,960-81,920 320,000-640,000
TABLE AMPLIFICATION
OF ~~~~~~~
ANTI-T2
Antiglobulin
1: 2ob
5 Rabbit received lo9 PFU T2 IV on day 0. b Rat anti-rabbit globulin, final dilution 1:20. c See Fig. 1. d Rabbit, immunized intensively by repeated injections
8 30 267
Complement”
S-16 256 128-256 128 256 8
lli
mi Ii-l-l'h 3‘2 i--e 4
of T2.
6
NEUTRALIZING
ACTIVITY
BY AKTIRABBIT
Antiallotype Rabbita
GLOI~I-LIV
ALLOTTPL
serumb
---~
Allotype
1,1/4,4 3,3/5,5
2,2/4,4
None
1
3
160c 160-320 320
10,240 640 640
0 Rabbit, of allotype shown, bled 11 days after lo9 PFU b Rabbit immune sera specific for allot,ypic determinants c 507, neutralization titer.
min at 100,000 y, but t’his operation also led to variable losses of antibody from the supernatant fluid. Shown in Table 5 are t,he data from a series of titrations in which a rat antiserum to rabbit r-globulin was used in what was established to be its maximally effective dose. This reagent proved to be highly active in that it amplified the neutralizing activities of sera obtained on days 6-35 by a factor of 128-256. It is noteworthy that it,, like complement, failed to amplify the activity of the earliest antibody (day 2) as effectively as that of 6-day antibody. Also of interest is the finding that the antiglobulin reagent still enhanced with peak efficiency the neutralizing act’ivity of sera obtained on days 21 and 35 while complement failed to do so. However, when applied to hyperimmune antibody both reagents had only minor and similar effects.
4 10,240
5120-10,240 320
320 10,240
~I:IU
-
5
6 _-
160 10,240 32OiXO
160
-~ --
T2 IV. al, a3, b4, b5, or b6, respectively.
Enhancement
by Antiallotype
Sera
In a series of experiment,s antiallotype sera were substituted for the antiglobulin serum described above. The results, summarized in Table 6, show that highly effective and specific amplification was attained. The appropriate antiallotype serum, whether it was directed against heavy or light polypeptide chain determinants, enhanced the neutralizing activity of sera from rabbits immunized against T2 phage 11 days earlier approximately 64-fold. This observation appears of particular interest because the allotypic determinants in question are expressed in the variable portions of the two polypeptide chains, thus in that part of the immunoglobulin molecule which also bears the antibody combining group. DISCIJSSIOK
The data presented in this paper document the maturation of neutralizing activity in
804
ADLER,
WALKER,
anti-T2 phage immune sera from dependence upon auxiliary serum factors early after immunization to a later dominance of antibody that is independent of such factors. The diverse enhancing agents, namely complement, heterologous antiglobulin serum, and antiallotype sera all closely resemble each other with regard to the extent of amplification t)hat they provide. The sole divergence observed is the more rapid decline of complement mediated enhancement which occurs during the third and fourth weeks of the antibody response, a time at which antiglobulin enhancement is still quite effective (Table 5). While we shall return to a discussion of this point later, it seems important at this time to call attention to the observation that the kinetics of the early antibody response, measured by neutralizat’ion test procedures in t,he presence of amplifying factors are quite different from those measured in their absence (Fig. 1) but parallel closely the kinetics of the antiT2 response as determined by passive hemagglutination (Walker et al., 1971). This suggests that the several amplifying factors may function by conferring neutralizing activity upon antibody which is intrinsically ineffective in this property. The broad biological implications of the amplifying mechanism have been cogently discussed in recent papers (Linscott and Levinson, 1969; Daniels et al., 1970), but two major points deserve reemphasis: the ability of complement to enhance neutralization lOO-lOOO-fold during the early phases of the still feeble antibody responseappears to be not only an ingenious and effective protective device of nature but also suggests one of the relatively few clearly defined biological roles for complement. Second, in vitro assays designed to measure the efficacy of vaccines, or diagnostic tests that depend upon the detection of changes in neutralizing activity might yield grossly misleading results if the differential in enhanceability of early and late antibody is ignored. It must be stressed, however, that the dependence of early antibody upon amplifying factors has thus far been demonstrated only in the limited number of virus-ant’i-
AND
FISHMAN
body systems cited earlier in this paper. A notable exception is antibody produced against the E. coli phage $X174, as shown in careful studies of H&jek (1970). Interestingly enough, if T2 phage to which certain haptens have been coupled is used to measure the kinetics of an ant,ibodv responseagainst the hapten, data are obtained which suggest that even t’he earliest detectable antibody neutralizes such phage effectively without added serum factors (Haimovich and Sela, 1969; Sarvas and M&kel%, 1970). Finally, although we lack systematic data on this point, it seemsreasonable to suppose that immunization with a larger dose of antigen than that used in the present immunization might hasten maturation of the immune responseand obscure the phase of dependence upon amplifying factors. Among t)he several possible mechanismsof neutralization enhancement, a direct virucida1 effect has been ruled out experimentally (Muschel and Toussaint, 1962) and seems improbable, a priori, in view of the similarities of antiglobulin and complement in their enhancing activities. It has also been suggestedthat antiglobulin and complement may act by adding bulk to the virus-antibody complex and thus further hinder adsorption to or penetration of the host cell. We suggest that this is not likely to be the correct explanation becauselimiting amounts of “late” antibody should then be equally suscept,ible to amplification of their neutralizing activity, yet they clearly are not. Two other possible explanations deserve att’ention. One of these is suggested by the data (Tables 3 and 4) which show that complement prevents dissociation of virusantibody complexes and thus reinforces selectively the neutralizing activity of early antibody which has a greater intrinsic tendency to dissociate than late antibody (Eisen and Siskind, 1964). The other possible mechanism, to which we have already alluded, rests on considerations of the antigenic and topographical complexity of T2 phages and the corresponding diversity of the specificities of anti-T2 antibodies. It is generally accepted that antibodies against head proteins neutralize phage much less efficiently than do
AMPLIFIED
so.-,
NEUTHALIZATTOX
antibodies against tail fiber antigens. It is conceivable that during the early phases of the immune response the fromation of ant,ibodies reactive with head antigens is dominant over the synt#hesis of antibodies to tail antigens. Thus the apparently preferentrial amplificatjion of early antibody activity may be more closely related to the specificit’y of such antibodies than to other att’ributes, such as class or avidit,y. If t)here is merit to f’his suggestion which, of course, is susccpt’ible to experimental proof, one is tempted lo speculate that antibodies to viruses which possess a simpler antigenic topography than T2 phage may not display the phenomenon of “maturation” from dependence to independence with regard to amplifying factors. The haptenated phage and its reaction with antibody against the hapten, discussed above, may be a contrived but nevert’heless valid model of a virus that is “simple” with regard to it’s exposed antigenic determinant’s. Our observation that, highly effective amplificat’ion by antiglobulin persists longer than that by complement (Table 5) suggests that’ 110 single proposed mechanism will adequately explain all facets of enhanced neut’ralization. While ant.iglobulin and antiallotype reagents clearly must act through attachment to a relatively fixed structural feature of the neutralizing antibody, i.e., it’s antigenic determinants, complement action depends upon the variable intrinsic ability of antibodies to f?x complement’, a property which furthermore depends on appropriate spa.cing of antibodies on the ant’igen. With reference to the action of antiglobulin, a. minor difference exists between our data and those of Hampar et al. (1968). While they failed to observe enhancement of neutralization by IgM antibodies, we saw highly significant’ amplificat)ion. We suggest I hat this may simply rcflrct the fact that our reagent was rich in ant i-w and a&-L chain antibodies. In experiments involving other antigen-antibody reagent)s it could be shown to have strong antiglobulin activity against both rabbit Igptl and IgG antibodies before absorption with IgG, and good residual act’ivity solely against, Ig&I after such absorption.
Last, a comment on the amplifying I(:t’ivity of antiallotype sera is indicatr>tl. rThe amino acid residues responsible t’o allot,ypic markers on heavy or light chains are believed to be located in the variable portion of these chains (Koshland, 1967). It is somewhat surprising, thercforcx, 1o note that the allot’ypic determinant’s obviously are not, blocked by the combinat,ion of I he antibody combining group, also in the variable portion, with a large antigen, such as T2 phage. The data of Reisfeld (1967J, who has shown that det#erminants reactive i\it h antibodies against b4 reside in several peptides derived from b4 light polypeptidn chains, suggest, that possibly some, but not, all, of the multiple determinants that, best 011 b4 specificit#y are sufficiently remoto from t hc antibody combining site to remain accessible after this site has been blocked. This observation is in full accord with the findings of Spring et al. (1970), who have shown that, specific binding of hapten to antibody does not, block the receptors for antiallotypn antibodies against “a” or “b” specificities. In the dat’a reported by us it is shown t IMI; even a macromolecular antigen that has combined with antibody fails to block sucsh receptors. It is of some interest th:11 the idiotypic determinants on ant ibody moltcules also seem to remain accbessiblc :lftrr such antibodies have combined wil h antigen (Kelus and Gel], 1968). This may not. IX universally correct), however, and may ~VCII have a t,rivial explanation as shown in recent studies of others (Brient, and Xisonoff. 1970).
We wish to thank Meredith Stokes, Susan Bit,tker and Raymond Harris for their technical assistance, and Katherine Kelly for her help in pre~~aring the manuscript. Rl?FlXHENCE:S ADLER, F. L., FISHMBN, M., and DRAY, 8. (1966). Antibody formation init,iated in vitro. III. Antibody formation and allotypic specificity directed by ribonucleic acid from peritoneal ~YIIdate cells. J. Immunol. 97, 554-558. ANDREWES, C. I-I., and ELFORD, W. J. (1933). Observations on antiphage sera: I. “The Percentage Law.” B,iC. J. Erp. Pathol. J4, 3(i7-:(7(i.
806
ADLER,
WALKER,
1~. W., and NISONOFF, A. (1970). Quantitative investigation of idiotypio antibodies. IV. Inhibition by specific haptens of the reaction of anti-hapten antibody with its anti-idiotypic antibody. J. Esp. Med. 132, 951-962. COWAN, K. M. (1962). Studies on the coIiphage neutralizing activity of normal human serum. BRIENT,
J. Immunol. 88, 476-481. DANIEI;S, C. A., BORSOS, T., RAPP, H. MAN, R., and NOTKINS, A. L. (1970).
J., SNYDERNeutralieation of sensitized virus by purified components of complement. Proc. Nat. Acad. Sci. U. S. 65, 528-535. EISEN, H. N., and SISKIND, G. W. (1964). Variations in affinities of antibodies during the immune response. 3ioc~emistry 3, 9~1~8. GINSBERG, H., and HORSFALL, F. L. (1949). A labile component of normal serum which combines with various viruses. Neutralization of infectivity and inhibition of hemagglutination by the component. J. Exp. Med. 90,475-495. HAIMOVICH, J., and SELA, M. (1969). Proteinbacteriophage conjugates: Application in detection in detection of antibodies and antigens. Science 164, 1279-1280. IIAJEK, P. (1969). Neutralization of bacterial viruses by antibodies of young animals. I. Dependence of neutralizing activity of 19 S and 7 S antibodies on complement in the course of primary and secondary response of young animals immunized with T2 phage. Folia Microbial. (Prague) 14, 165-170. IUJEK, P. (1970). Neutralization of bacterial viruses by antibodies of young animals. II. The development of the avidity of 19 S and 7 S neutralizing antibodies in the course of primary and secondary response in young rabbits immunized with +X 174 bacteriophage. Folia Microbial. (Prague) 15, 9-16. HAMPAR, B., NOTKINS, A. L., MAGE, M., and KEEHN, M. A. (1968). Heterogeneity in the properties of 7 S and 19 S rabbit-neutralizing antibodies to herpes simplex virus. J. Imm~~ol. 199, 586-593. HEINEMAN, H. S. (1967). Herpes simplex neutralizing antibody-quantitation of the complementdependent fraction in different phases of adult human infection. J. Immunol. 99, 214-222. ISHIZABA, K., and CAMPBELL, D. II. (1959). Biologic activity of soluble antigen-antibody complexes. V. Change of optical rotation by the formation of skin reactive complexes. J. Immunol. 83, 318-326. JERNE, N. K., and AVEGNO, P. J. (1956). The development of the phage-inactivation properties of serum during the course of specific immunization of an animal: reversible and irreversible inactivation. J. Immunol. 76, 200-208.
AND FISHMAN A. S., and GELL, P. G. H. (1968). Immunological analysis of rabbit anti-antibody systems. J. Exp. Med. 127, 215-234. KOYHLAND, M. E. (1967). Location of specificity and allotypie amino acid residues in antibody Fd fragments. Cold SpT~ng Harbor Sop. count. Biol. 32, 119-127. LAFFERTY, K. J. (1963). The interaction between virus and antibody. II. Mechanism of the reaction. Virology 21, 76-90. LINSCOTT, W. D., and LEVINSON, W. E. (1969). Complement components required for virus neutralization by early immunoglobulin antibody. Proc. Nat. Acad. Sci. U. S. 64, 52@527. MUELLER, J. H. (1931). The effect of alexin in vied-antivirus mixtures. J. ~mmunol. 26,17-23. MUSCHEL, L. H., and TOUSSAINT, A. J. (1962). Studies on the bacteriophage neutralizing activity of serums. II. Comparisons of normal and immune phage neutralizing antibodies. J. Immunol. 89, 35-40. OZAKI, Y., and TABEYI, K. (1967). Studies on the neutralization of Japanese encephahtis virus. I. Application of kinetic neutr~ization t.o the measurement of the neutralizing potency of antiserum. J. Immunol. 98, 12181223. REISFELD, R. A. (1967). Heterogeneity of rabbit light-polypeptide chains. Cold Spring Harbor Symp. Quant. Biol. 32, 291-298. SARVAS, H., and MBKE& 0. (1970). Haptenated bacteriophage in the assay of antibody quantity and aflinity: maturation of an immune response. Irnrn~~oc~~i~tr~ 7, 933-943. S~ORDINTSEV, A. A., and YABROW, A. A. (1963). Intensity of the interaction of antibody and viruses in the haemagglutination inhibition test in the presence of non-specific thermolabile stimulator of antibodies. Acta Viral. (Prague) 7, 138-151. SPRING, S. B., NISONOFF, A., and DRAY, S. (1970). Independence of allotypic determinants and antibody specificity. J. Immuno~. 105,653-660. STYK, B. (1962). Cofactor and specific antibodies against influenza viruses. III. The potentiating effect of cofactor on specific antibodies of early immune and hyperimmune sera and the differences in the character of these antibodies. Acta Virol. (Prague) 6, 327-337. SVEHAO, S.-E., ~~~BLoTH, B. (1967). Examination of virus-yM antibody complexes by electron microscopy. I. Confi~ration of -&% antibodies and mode of attachment to antigen. Virology 31, 676-687. TOWSSALNT, A. J., and MUSCHBL, L. H. (1962). Studies on the bacteriophage neutralizing activity of serums. I. An assay procedure for normal antibody and complement. J. Immtinol. 89, 27-34. KELUS,
AMPLIFIED
NEUTRALIZATION
WALKER, W. S., LIU, C. T., and ADLER, F. L. (1969). The detection of anti-phage antibodies by passive hemagglutination. J. lmmunol. 103, 907-916. W.~LKER, W. S., FISHMAN, M., and ADLER, F. L. (1971). Detection of anti-phage antibodies by passive hemagglutination. II. Early antibodies produced in vivo and in vitro. J. Immunol. 107, 953%95ci.
m’i
WAY, H. J., and GARWES, D. J. (1970). Serum accessory factors in the measurement of arbovirus neutralization reactions. J. Gen. Viirol. 7, 211223. YOSHINO, K., and TANIGUCHI, S. (1965). Studies on the neutralization of herpes simplex virus. I. Appearance of neutralizing antibodies having different grades of complement reqllirement. Virology 26, 44-53.
46, 797-807
(1971j
Amplification
of AntiGlobulin,
l?. L. ADLER,
Phage
Neutralization and
by
AntiAllotype
W. S. WALKER,’
Complement, Sera’
.\NI) RI. FISHMAN
Rabbits and rats injected once with lo9 PFU of coliphage T2 (0.1 ,~g nitrogen) I’+ spend with neutralizing antibody which during the first 2 weeks is minimally efficient. Complement, antiglobulin, or antiallotype antibody amplify the activity of such early IgM and IgG antibodies up to 1000.fold. and the kinetics of the early antibod? response measured in the presence of these enhancing reagents closely resemble each other but differ from the kinetics of the neutralizing antibody response determined irr t,he conventional manner. 1IVTROI)UCTIOIV
t*he course of our studies on t,he induction of antibody formation against T2 bacteriophage in vitro, the need for highly sensitive assays attuned to the detection of “early” antibodies arose. A passive hemagglutination assay n-as developed which filled this requirement8in part (Walker et al., 1969, 1971), and a neutralization test that employs complement or antiglobulin as amplifying agents has been explored. A role for complement’ or some of its component)sin the nrutralization of viruses by nnt,ibody has received sporadic at,tention in the litcruturc wer since J. H. Mueller’s observation (1931) on the role of “alexin” in the neut,ralization of vaccinia virus. Ginsberg and Horsfall (1949) found that’ calcium ion and thcrmolabilc serum components contributed to the neutralization of myxoviruses by normal sera. It was shown subsequently that dwomplcmc~ntation removed the IWUlrnlizing activity of normal swa for the T In
’ This work was supported in part by (+rant AI-06890 from the Xational Institute of Allergy and Infert,ious Iliseases, and by Grant GH-29018X from the National Srience Foundation. * Supported by Training Grant AI-00408 from the National Institute of Allergy and Infectious Diseases. 797
phages of Escherichia coli (Cowtrl, 1962), and t’hat both complement and specific* antibody were required (Toussaint and Muschcl, 1962; Muschel and Toussaint, 1962). Antibody appearing early in immunization, like normal antibody, was shown to depend on complement, not only in t I](. neutralizat’ion of T phagcs (~lus~l~~~land Toussnint, 1962; HBjek, 1969), but also in the inactivation of myxoviruscs (St,yk, 1962; Smordintsw and ‘L’abrov, 1963; Lirwot t and Lrvinson, 1969), hrrpcs simpltlx virus (Yoshino and Taniguchi, 196n; Hcineman, 1967; Hnmpar et al., 1968) and arboviruws (Ozaki :md Tabrbyi, 1967; Way and Gnrnw, 1970). The neutralizing activity of Ig1I as w11 as that of “early” IgG ant,ibody is c~nhanwtl by wmplcment (Yoshino and Tanigwhi, 1965; Hampar et al., 196X; H&j&, 1969), and rewnt worlc has shown that only t h(k first’ wmponents of complement are required for amplification (Linscot t and LcvinscIII, 1969; Daniels ebal., 1970). Another series of rcccnt papers prwwfs data on the neutralization of infectiw complexes of virus-ant’ibody by antiglobulin reagclnts (Hampar et al., 1968). The data to be prescntc>dconfirm and cxxtclnd thrw findings.
798
ADLER,
WALKER,
AND
FISHMAN
The antiglobulin used in most experiments was prepared by intensive immunization of Immune sera. Concentrates of coliphage rats with a fraction of rabbit serum globulin T2 (2 X 1012 PFU/ml) were prepared as rich in IgM (first peak from Sephadex Gpreviously described (Walker et al., 1969), 150). Precipitin analysis in agar gel showed and rabbits and rats were injected intravethis serum to react both with purified IgG nously or intraperitoneally with lo9 PFU and IgM fractions. After absorption with an (0.1 pg nitrogen) in 1.0 ml. Selected immune excess of IgG it still precipitated IgM, sugsera were decomplemented by incubation gesting that it contained antibodies of anti-k with preformed immune complexes of bovine specificity as well as antibodies to light serum albumin (BSA) and rabbit anti-BSA polypeptide chains shared by IgG and IgJ1. formed at equivalence. Amplification by antiallotype serum. The Complement. Pooled sera from normal rats procedure was exactly the same as that used selected for low “background” anti-T2 neufor antiglobulin. Reagents used in the prestralizing antibodies were used in fmal diluent investigation were prepared by intensive tions of 1: 20 or 1:40. For experiments immunization of appropriate rabbits wit’h requiring higher concentrations of complerabbit r-globulin. The anti-4 reagent was ment, sera were obtained from rats subjected made in al,al/b5,b5 rabbits by injection of to whole body X-irradiation (650 R) and al,al/b4,b4 globulin, the anti-6 reagent by bled 5 days later. Individual sera from such globulin into al,a3/ animals were screened for their lack of sig- injecting al,u3/b6,b6 b5,b5 rabbits and the anti-5 serum by immunificant T2 neutralizing activity in a final nization of al,al/b4,b4 rabbits with al,al/ dilution of 1: 10, as well as for their ability b&b5 globulin. The anti-l (053P) and anti-3 to amplify an early anti-T2 complement(E-275-6) sera were kindly donated by Dr. dependent immune serum. Appropriate sera were pooled and stored in small volumes at Rose Mage. All were potent sera and were used after heat-inactivation (56” for 30 min) -70”. Rabbit sera, obtained 5 days after and lo-fold dilution. whole-body X-irradiation of 1000 R, were found to be as effective as rat sera in enhancRESULTS ing complement-dependent neutralizing activity. In all instances, the dose of compleComplement Dependence of Early Antibody ment used in the assay of antibody was after a Single Injection of Phage chosen so that no more than 10 % of the test A group of 12 rabbits each received a dose of phage was neutralized by complesingle intravenous injection of lo9 PFU of ment alone. T2. Serial bleedings were titrated for their AmpliJication by complement. Serially di- neutralizing activity with or without the luted antisera or tissue culture fluids, in addition of complement, with the resulbs 0.4~ml amounts, were incubated for 30 min shown in Fig. 1. It will be noted that 50% at 37” after addition of 0.2 ml of suitably neutralization titers NTE~ measured in the diluted complement and 0.2 ml of a phage absence of added complement rose, at a suspension diluted to contain 400 PFU of T2. moderate rate, to a mean of 160 on day 11 Duplicate or triplicate aliquots of 0.2 ml and then showed no further significant were then plated on E. coli B. Further de- increase to day 35. In contrast, titrations in tails are described in the text. the presence of added complement showed a AmplQication by antiglobulin. Serial dilurapid rise in activity which attained a peak tions of antiserum or tissue culture fluid of 10,240-20,480 on day 5 or 6, maintained (0.4 ml) were incubated with 0.2 ml of phage itself at that level until about day 15-20 suspension (400 PFU) for 30 min at 37” and then declined. The most effective amfollowed by a second incubation period of plification by complement occurred in the 90 min at 4”. A potent antiglobulin serum bleedings obtained between days 4 and 15 (heated 56” for 30 min) was then added (0.4 when titers were 128-256 fold greater in ml of 1: lo), and after an incubation for 30 the presence than in the absence of added min at 37” aliquots were plated as above. complement (0.2 ml 1: 10). Thereafter the MATERIALS
AND
METHODS
AMPLIFIED
NEUTRAI,IZ,1TTON
TR!)
amplification was less; on day 35 it merely averaged 4-fold. Hyperimmune sera (Walker et al., 1969) showed a similarly small amplification of their neutralizing act,ivities. The da$a just preser&ed suggested that neutralizing activity of the earliest antibody, like that of the antibody found aft(er day 15, was not as effect’ively amplified by complement as that of the antibody found in the intervening period. The data presented in Fig. 2 show that the amount of complement used in the previous experiments raised t,he NT&o of a Z-day bleeding from 32 t,o 128, 5 mere $-fold rise. By increasing the complement dose 16-fold amplification was obtained. Decomplementation of the serum, on the other hand, led to a, 4-fold decrease of NT50, Since the change in regard to complement amplification appeared to be within the IgM class of antibodies, as will be discussed in the next section, it appeared of interest to determine whether “late” IgM antibody resembles t’hat found on day 2 or that found during the period of maximally enhanceable IgM antibody. A purified IgM fraction from a al-day bleeding was examined, and it was found that its neut,ralizing activity was ampli~ed 2~0-fold by complement. It appears, therefore, that IgM antibody formed between day 4 and approximately day 15 is enhanceable by complement to a significantly greater degree than IgM formed before or after this period. Changes in specificity, affinity, or in complement fixing ability may be responsible singly or in combination, and furt.her work is needed to resolve this problem.
at OD 250 nm. A pool of fractions 3-7 was designated IgM, and fractions 11.-U ant1 14-17 were pooled to yield IgG prepnra tions I and II. To confirm the ahsencc of IgM ant,ibody in the t,wo IgG prepar~~tiol~s, :I pool of fractions 11-17 was subject,ed to a second centrifugation in a sucrose gradient. Fractions l&l5 from this gradient ~ontainetl neutralization activity (7 S) I\-hicall was readily enhanced by complement. As shown by the data in Table 1, both IgM and IgG antibody from this serum was an~plified in its neutralizing activity, the IgM antibody more effectively than IgG. These data, t,ogether wit’h those shown in Figs. 1 and 2, are in agreement with the results of others (H&jek, 1969) and show Ohnt while IgM antibody present on day 11 (days 4-15) is most readily amplified, the IgG ant*ibody found on day I1 is enhanced no less than the TgLM antibody found prior to day 4 and not si~ificantly less t,han IgM in the 2X-day bleeding.
ImmunogEobulin
COMPLEWENT AMPMFICXTION OF NEUTU \LIZATIOK IJY IgM AND IgG ANTIRODTIW --.- -. ~~-..
Class of Neutralizing
Anti-
body Enhanced by Complement
While little or no neutralizing IgG antibody could be found in sera obtained during the first week after immunization, the llday bleeding permitted a comparative study of IgM and IgG neutralizing antibodies with respect to complement amplification. The serum was placed on a lO-37% linear sucrose gradient and was subjected to 16-hr centrifugation at 32,000 rpm in a SW 50.1 Spinco rotor. The contents of each gradient tube were collected in 20 equal fractions. 6 and 17, respectively, had absorption peaks
Titration of Complementfor Amplifying tivity
.-1c-
If the technique of complement-ampl.ified neutralization is to yield meaningful results as a measure of antibod~T activity, it is is important to ascertain that antibody and not t)he accessory factors determine the end point,. The presence of varying amounts of normal antibody reactive with T2 phage in the sera of nonimmunized rats imposed an upper limit on the amount of complement that could be used without complicating TABLE
50% Globulin classa
IgM IgG IgG
pool pool pool (L Sucrose
I IL
1
-
Neutralization _-i
jcomppment
/ C;;$-
fractions
.-._
;im$l?p-
!-.--_-.-_---_~__-^! / 1632 i 2048-4~~ 4-x / 128 I j 4-8 128 gradient
titer .-- .._ .I
128X 16-32X 16--32X . ..--.._-II-day bleed-
from
ing. 6 Complement, source: dilution 1:40 (25 ~.rl).
Normal
rat
scrltm,
final
800
ADLER,
WALKER,
AND
FISHMAN
. .
.
.
A
Y
l A
m l
0 l
FIG. 2. T2 neutralizing activity of a 4.day bleeding from a rabbit that received 109 PFU of T2. The serum was assay untreated (a), after decomplementation (0), decomplemented followed by the addition of rat complement, final dilution 1:40 (X) or rat complement from X-irradiated animals, final dilution 1:lO (A).
complement of extremely low “normal” antibody content, and it was possible to use such serum as complement in amounts of up to 200 /.ll. Figure 3 shows the composite results of complement titrations in which serial dilutions of a decomplemented rat antiserum to T2, obtained 4 days after the intraperitoneal injection of log PFU, were titrated in the the assay. By the use of pools of sera from presence of varying amounts of rat compleselected rats, this upper limit was 20-40 ~1 ment. The larger dosesof complement (25in the standard test as described in the 200 fil) were from a pool of sera from Xprevious figures and table. Several attempts irridiat,ed rats. It may be seenthat maximal were made to remove the “normal” phage- amplification (512-1024-fold) was att,ained neutralizing antibody from sera that were wiyith 100 ul of complement and that a to be used as complemenL Absorptions measurable degree of amplification required with phage that had been inactivated by only 1.25 ~1. It will also be noted that the irradiation or by treatment w&h formalin experiments already discussed were done in were carried out in the cold to preserve the presence of that amount of complement complement activit’y. It was found that (20 .ul) which represents the upper limit of such treatment frequently resulted in en- the linear portion of the plot shown in Fig. 3. hanced “neutralizing” activity which proved to be independent of complement and, on The Inhibitory E$ect of Phosphate Ions further examination, could be shown t)o be In titrating the sera from normal rats it a result of the introduction into the serum was found that t,hose with NTso of less of solubilized E. coli antigens. The use of than 4 when diluted in phosphate buffer sera from rats that had been subjected to (2.25 mM) often neutralized T2 phage tot’al body X-irradiation (650 R) provided when they were diluted 44Sfold in buffer FIG. 1. T2 neutralizing
activity of rabbit sera following a single intravenous injection of lo9 PFU T2 on day 0. Each symbol stands for the titer of 1 rabbit serum obtained on the day indicated. Assayed in the absence (0) or presence (A) of 0.02 ml of active normal rat serum as complement source.
AMPLIFIED
SO1
NEUTIIALIZATIC)PU’ TABLE
PhosphateY (m&0 _.---_-
2
Percent neutralization bv ran anti-T2 antibody ifinal dilution ib __--~-.__ -..-~~ ~-.-... 1:250 1:12.50 1: 6250 _____ -----.--. ~_-.~ ..-.__ - -
99 98 30 96 R-2 !N 100 !).i ::i !I.-> 3X 9') IN 96 3I 97 87 2.i 90 51 7 I!) II 80 _--~..__ ---.Q Phosphate buffer, pH i.3. * Each reaction mixtllre (0.8 ml) contained 0.4 ml of diluted rat anti-T2 serum, 0.2 ml of rat complement 1: 10, and 0.2 ml phase suspension. The Mg2+ and Ca2+ were 0.25 m&’ and 0.075 m.W. wspectively. 0 0.75 1.5 3.0 6.0 12.0 24.0 37.5
3. Neutralizing activity of a decomplemented rat antiserum obtained 4 days after int,raperitoneal injection of 109 PFU of T2. Assayed in the presence of increasing amounts of rat complement. FIG.
lacking phosphate and containing magnesium and calcium (0.5 and 0.15 &I’, respect,ively). To investigat’e the apparent dependence of complement-enhanced neut’ralizing activity on suitable Ca2+ and lMg2+ concentraGons, and to ascertain the antagonistic effect of PO&, serum obtained from rats 4 days after a single injection of lo9 PFU of phage was titrated with complement,, Mg2;2f and CM?+ at, constant concent’rations while POa2- and antibody were varied. The results, summarized in Table 2, showed that under the condit,ions chosen PO?- concentrations of 12 n& or higher inhibited si~ificantl~ and that this effect was most pronounced when limiting amounts of antibody were approached. The NT60 of the serum, in the absence of added complemerit, was 40-80. It is clear, therefore, that the inhibitory effect of POF, although marked, was not absolute within the conwntration range tested.
The enhanced rwuralization of rort~ain viruses by antibody in the presence 11f complement has been attributed to the effect of the added bulk of the ahsorbtrd protein on absorption or penetration of the virus in its reaet,ion -tvith cells (Daniels ct ui., 1970; Linscott and Levinson, 1969j. Wc considered alternate or additional mwhanisms, such as possible effects of cornpI+ ment on the rate of conformational changc~s in antibody that occur secondarily to its reaction v&h the virus (Jshizaka and Campbell, 1959; Laffert-y, 1963; Svehag and Blotlr, 1967 and others), or on the ~socia~i{)~~ rate of virus and antibody. I’re~irnirI~~r~ experiments yielded results which illustrated certain difficulties in applying convent ion aI kinetic analysis procedures to L4t~arly” mtibodies. Summarized in Table 3 are data obt:rined with a 5 day bleeding from a rabbit3 immuniz~ with T2. To allow comparisor~~ of the degree of Ileut,ra~izatio~l of phage plat cd dire&Iv with results obt,ained by plating aft>er dilution, the range of phage conwntrations was restricted to S-fold. It will be noted that the neutralization reaction closely followed the “percentage law” (Andrcws and Elford, 1933) except -\l-hen plating wts
802
ADLER,
WALKER,
AND
TABLE REACTIVATION
FISHMAN
3
OF NEUTRALIZED
VIRUS
BY DILUTIOLY
Phage input Anti-T2 serum=
1:20 1:40 1:80
976
2% Undil.”
II%.
le (1OO)f 13 (95) 76 (70)
8 (99) 33 (93) 109 (77)
1952
Undil.
Di1.d
Undil.
Dil.
13 (99) 60 (94) 223 (77)
32 (96) 176 (82) 520 (47)
29 (99) 140 (93) TMTCQ
269 (85) 664 (66) 1040 (47)
a Rabbit serum (5 day) following log PFU T2 IV. Values given are final dilutions. b Total number of phage added to incubation mixtures. c Plated without dilution. d Diluted prior to plating. e Total PFU in volume plates. f Values in parentheses are percent neutralization of phage input. 0 Too many to count. TABLE EFFECT
OF COMPLEMENT OF NEUTRALIZED
4
ON VIRUS
THE REACTIVATION BY DILUTION
I
Total PFU T2 added 840 1 8,404 1 84,000 1840,000
~-
1:20 1:40 1:80 1:4000 1:8000
+ + + + +
82b (34)” 100 100 74 (28) 100 100 99 37 (12) 100 99 100 98 93 (62) 89 91 (30)
(47)
(34)
(& (84) 04) (82) (50)
(& (82) (20) (83) (23)
(12) (19)
0 Rabbit serum obtained 5 days after intravenous injection of 109 PFU T2. Values shown are final dilutions. b Percent neutralization in sample plated without dilution. c Values in parentheses are percent neutralization in sample diluted lo-fold or more before plating.
preceded by dilution. If the dilution step was interposed, marked reactivation took place unless the antibody concentration was very high (antiserum 1: 20). To test the effect of complement upon dissociation by dilution, a number of experiments was performed which yielded data of which those in Table 4 are representative. It is apparent from these data that dilution prior to plating (lo-fold or more) brought about marked reactivation of “neutralized
virus” from reaction mixtures, even from those which contained high levels of antibody but no complement. The data further show that the reactivation by dilution affected a fixed percentage of “neutralized virus” over a 1006fold range of virus concentrations. In the presence of complement and high antibody concentration, there was no dissociation upon dilution. When antibody approached limiting concentrations, complement no longer prevented dissociation upon dilution. Complement, therefore, appears to reinforce the dissociable bond between virus and antibody. Comparative Studies on Amplification by Complementand Antiglobulin Since antiglobulin has been successfully employed in amplifying the neutralizing activity of antisera against certain viruses (Hampar et al., 19SS), leading to the suggestion that this reagent acts like complement by adding bulk to the virus-antibody complex, some experiments were done to compare these reagents. An added reason was provided by the unfortunate presence of high levels of anticomplementary activity in tissue culture fluids from cultures in which antibody against T2 phage had been induced in vitro by the method previously described (Adler et al., 1966). This activity, often still present in fluids diluted 8-16-fold, appeared to be concentrated in particulate material which could be spun down in 90
AMPLIFIED
NEUTRALIZATION TABLE
-~
AMPLIFKXTION
OF RABBIT
ANTI-%!
50% Bleedinga
5
NEUTKALIZISG
Neutralization
SO:<
ACTIVITT
BY AXTIIUBBIT
Amplification
titer
(day) No antiglobulin 2
Antiglobulin
40 160 160 160-320 160-320 40,000-80,000
6
15 21 35 Hype@
320-640 40,960 20,48040,960 20,480-40,960 40,960-81,920 320,000-640,000
TABLE AMPLIFICATION
OF ~~~~~~~
ANTI-T2
Antiglobulin
1: 2ob
5 Rabbit received lo9 PFU T2 IV on day 0. b Rat anti-rabbit globulin, final dilution 1:20. c See Fig. 1. d Rabbit, immunized intensively by repeated injections
8 30 267
Complement”
S-16 256 128-256 128 256 8
lli
mi Ii-l-l'h 3‘2 i--e 4
of T2.
6
NEUTRALIZING
ACTIVITY
BY AKTIRABBIT
Antiallotype Rabbita
GLOI~I-LIV
ALLOTTPL
serumb
---~
Allotype
1,1/4,4 3,3/5,5
2,2/4,4
None
1
3
160c 160-320 320
10,240 640 640
0 Rabbit, of allotype shown, bled 11 days after lo9 PFU b Rabbit immune sera specific for allot,ypic determinants c 507, neutralization titer.
min at 100,000 y, but t’his operation also led to variable losses of antibody from the supernatant fluid. Shown in Table 5 are t,he data from a series of titrations in which a rat antiserum to rabbit r-globulin was used in what was established to be its maximally effective dose. This reagent proved to be highly active in that it amplified the neutralizing activities of sera obtained on days 6-35 by a factor of 128-256. It is noteworthy that it,, like complement, failed to amplify the activity of the earliest antibody (day 2) as effectively as that of 6-day antibody. Also of interest is the finding that the antiglobulin reagent still enhanced with peak efficiency the neutralizing act’ivity of sera obtained on days 21 and 35 while complement failed to do so. However, when applied to hyperimmune antibody both reagents had only minor and similar effects.
4 10,240
5120-10,240 320
320 10,240
~I:IU
-
5
6 _-
160 10,240 32OiXO
160
-~ --
T2 IV. al, a3, b4, b5, or b6, respectively.
Enhancement
by Antiallotype
Sera
In a series of experiment,s antiallotype sera were substituted for the antiglobulin serum described above. The results, summarized in Table 6, show that highly effective and specific amplification was attained. The appropriate antiallotype serum, whether it was directed against heavy or light polypeptide chain determinants, enhanced the neutralizing activity of sera from rabbits immunized against T2 phage 11 days earlier approximately 64-fold. This observation appears of particular interest because the allotypic determinants in question are expressed in the variable portions of the two polypeptide chains, thus in that part of the immunoglobulin molecule which also bears the antibody combining group. DISCIJSSIOK
The data presented in this paper document the maturation of neutralizing activity in
804
ADLER,
WALKER,
anti-T2 phage immune sera from dependence upon auxiliary serum factors early after immunization to a later dominance of antibody that is independent of such factors. The diverse enhancing agents, namely complement, heterologous antiglobulin serum, and antiallotype sera all closely resemble each other with regard to the extent of amplification t)hat they provide. The sole divergence observed is the more rapid decline of complement mediated enhancement which occurs during the third and fourth weeks of the antibody response, a time at which antiglobulin enhancement is still quite effective (Table 5). While we shall return to a discussion of this point later, it seems important at this time to call attention to the observation that the kinetics of the early antibody response, measured by neutralizat’ion test procedures in t,he presence of amplifying factors are quite different from those measured in their absence (Fig. 1) but parallel closely the kinetics of the antiT2 response as determined by passive hemagglutination (Walker et al., 1971). This suggests that the several amplifying factors may function by conferring neutralizing activity upon antibody which is intrinsically ineffective in this property. The broad biological implications of the amplifying mechanism have been cogently discussed in recent papers (Linscott and Levinson, 1969; Daniels et al., 1970), but two major points deserve reemphasis: the ability of complement to enhance neutralization lOO-lOOO-fold during the early phases of the still feeble antibody responseappears to be not only an ingenious and effective protective device of nature but also suggests one of the relatively few clearly defined biological roles for complement. Second, in vitro assays designed to measure the efficacy of vaccines, or diagnostic tests that depend upon the detection of changes in neutralizing activity might yield grossly misleading results if the differential in enhanceability of early and late antibody is ignored. It must be stressed, however, that the dependence of early antibody upon amplifying factors has thus far been demonstrated only in the limited number of virus-ant’i-
AND
FISHMAN
body systems cited earlier in this paper. A notable exception is antibody produced against the E. coli phage $X174, as shown in careful studies of H&jek (1970). Interestingly enough, if T2 phage to which certain haptens have been coupled is used to measure the kinetics of an ant,ibodv responseagainst the hapten, data are obtained which suggest that even t’he earliest detectable antibody neutralizes such phage effectively without added serum factors (Haimovich and Sela, 1969; Sarvas and M&kel%, 1970). Finally, although we lack systematic data on this point, it seemsreasonable to suppose that immunization with a larger dose of antigen than that used in the present immunization might hasten maturation of the immune responseand obscure the phase of dependence upon amplifying factors. Among t)he several possible mechanismsof neutralization enhancement, a direct virucida1 effect has been ruled out experimentally (Muschel and Toussaint, 1962) and seems improbable, a priori, in view of the similarities of antiglobulin and complement in their enhancing activities. It has also been suggestedthat antiglobulin and complement may act by adding bulk to the virus-antibody complex and thus further hinder adsorption to or penetration of the host cell. We suggest that this is not likely to be the correct explanation becauselimiting amounts of “late” antibody should then be equally suscept,ible to amplification of their neutralizing activity, yet they clearly are not. Two other possible explanations deserve att’ention. One of these is suggested by the data (Tables 3 and 4) which show that complement prevents dissociation of virusantibody complexes and thus reinforces selectively the neutralizing activity of early antibody which has a greater intrinsic tendency to dissociate than late antibody (Eisen and Siskind, 1964). The other possible mechanism, to which we have already alluded, rests on considerations of the antigenic and topographical complexity of T2 phages and the corresponding diversity of the specificities of anti-T2 antibodies. It is generally accepted that antibodies against head proteins neutralize phage much less efficiently than do
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NEUTHALIZATTOX
antibodies against tail fiber antigens. It is conceivable that during the early phases of the immune response the fromation of ant,ibodies reactive with head antigens is dominant over the synt#hesis of antibodies to tail antigens. Thus the apparently preferentrial amplificatjion of early antibody activity may be more closely related to the specificit’y of such antibodies than to other att’ributes, such as class or avidit,y. If t)here is merit to f’his suggestion which, of course, is susccpt’ible to experimental proof, one is tempted lo speculate that antibodies to viruses which possess a simpler antigenic topography than T2 phage may not display the phenomenon of “maturation” from dependence to independence with regard to amplifying factors. The haptenated phage and its reaction with antibody against the hapten, discussed above, may be a contrived but nevert’heless valid model of a virus that is “simple” with regard to it’s exposed antigenic determinant’s. Our observation that, highly effective amplificat’ion by antiglobulin persists longer than that by complement (Table 5) suggests that’ 110 single proposed mechanism will adequately explain all facets of enhanced neut’ralization. While ant.iglobulin and antiallotype reagents clearly must act through attachment to a relatively fixed structural feature of the neutralizing antibody, i.e., it’s antigenic determinants, complement action depends upon the variable intrinsic ability of antibodies to f?x complement’, a property which furthermore depends on appropriate spa.cing of antibodies on the ant’igen. With reference to the action of antiglobulin, a. minor difference exists between our data and those of Hampar et al. (1968). While they failed to observe enhancement of neutralization by IgM antibodies, we saw highly significant’ amplificat)ion. We suggest I hat this may simply rcflrct the fact that our reagent was rich in ant i-w and a&-L chain antibodies. In experiments involving other antigen-antibody reagent)s it could be shown to have strong antiglobulin activity against both rabbit Igptl and IgG antibodies before absorption with IgG, and good residual act’ivity solely against, Ig&I after such absorption.
Last, a comment on the amplifying I(:t’ivity of antiallotype sera is indicatr>tl. rThe amino acid residues responsible t’o allot,ypic markers on heavy or light chains are believed to be located in the variable portion of these chains (Koshland, 1967). It is somewhat surprising, thercforcx, 1o note that the allot’ypic determinant’s obviously are not, blocked by the combinat,ion of I he antibody combining group, also in the variable portion, with a large antigen, such as T2 phage. The data of Reisfeld (1967J, who has shown that det#erminants reactive i\it h antibodies against b4 reside in several peptides derived from b4 light polypeptidn chains, suggest, that possibly some, but not, all, of the multiple determinants that, best 011 b4 specificit#y are sufficiently remoto from t hc antibody combining site to remain accessible after this site has been blocked. This observation is in full accord with the findings of Spring et al. (1970), who have shown that, specific binding of hapten to antibody does not, block the receptors for antiallotypn antibodies against “a” or “b” specificities. In the dat’a reported by us it is shown t IMI; even a macromolecular antigen that has combined with antibody fails to block sucsh receptors. It is of some interest th:11 the idiotypic determinants on ant ibody moltcules also seem to remain accbessiblc :lftrr such antibodies have combined wil h antigen (Kelus and Gel], 1968). This may not. IX universally correct), however, and may ~VCII have a t,rivial explanation as shown in recent studies of others (Brient, and Xisonoff. 1970).
We wish to thank Meredith Stokes, Susan Bit,tker and Raymond Harris for their technical assistance, and Katherine Kelly for her help in pre~~aring the manuscript. Rl?FlXHENCE:S ADLER, F. L., FISHMBN, M., and DRAY, 8. (1966). Antibody formation init,iated in vitro. III. Antibody formation and allotypic specificity directed by ribonucleic acid from peritoneal ~YIIdate cells. J. Immunol. 97, 554-558. ANDREWES, C. I-I., and ELFORD, W. J. (1933). Observations on antiphage sera: I. “The Percentage Law.” B,iC. J. Erp. Pathol. J4, 3(i7-:(7(i.
806
ADLER,
WALKER,
1~. W., and NISONOFF, A. (1970). Quantitative investigation of idiotypio antibodies. IV. Inhibition by specific haptens of the reaction of anti-hapten antibody with its anti-idiotypic antibody. J. Esp. Med. 132, 951-962. COWAN, K. M. (1962). Studies on the coIiphage neutralizing activity of normal human serum. BRIENT,
J. Immunol. 88, 476-481. DANIEI;S, C. A., BORSOS, T., RAPP, H. MAN, R., and NOTKINS, A. L. (1970).
J., SNYDERNeutralieation of sensitized virus by purified components of complement. Proc. Nat. Acad. Sci. U. S. 65, 528-535. EISEN, H. N., and SISKIND, G. W. (1964). Variations in affinities of antibodies during the immune response. 3ioc~emistry 3, 9~1~8. GINSBERG, H., and HORSFALL, F. L. (1949). A labile component of normal serum which combines with various viruses. Neutralization of infectivity and inhibition of hemagglutination by the component. J. Exp. Med. 90,475-495. HAIMOVICH, J., and SELA, M. (1969). Proteinbacteriophage conjugates: Application in detection in detection of antibodies and antigens. Science 164, 1279-1280. IIAJEK, P. (1969). Neutralization of bacterial viruses by antibodies of young animals. I. Dependence of neutralizing activity of 19 S and 7 S antibodies on complement in the course of primary and secondary response of young animals immunized with T2 phage. Folia Microbial. (Prague) 14, 165-170. IUJEK, P. (1970). Neutralization of bacterial viruses by antibodies of young animals. II. The development of the avidity of 19 S and 7 S neutralizing antibodies in the course of primary and secondary response in young rabbits immunized with +X 174 bacteriophage. Folia Microbial. (Prague) 15, 9-16. HAMPAR, B., NOTKINS, A. L., MAGE, M., and KEEHN, M. A. (1968). Heterogeneity in the properties of 7 S and 19 S rabbit-neutralizing antibodies to herpes simplex virus. J. Imm~~ol. 199, 586-593. HEINEMAN, H. S. (1967). Herpes simplex neutralizing antibody-quantitation of the complementdependent fraction in different phases of adult human infection. J. Immunol. 99, 214-222. ISHIZABA, K., and CAMPBELL, D. II. (1959). Biologic activity of soluble antigen-antibody complexes. V. Change of optical rotation by the formation of skin reactive complexes. J. Immunol. 83, 318-326. JERNE, N. K., and AVEGNO, P. J. (1956). The development of the phage-inactivation properties of serum during the course of specific immunization of an animal: reversible and irreversible inactivation. J. Immunol. 76, 200-208.
AND FISHMAN A. S., and GELL, P. G. H. (1968). Immunological analysis of rabbit anti-antibody systems. J. Exp. Med. 127, 215-234. KOYHLAND, M. E. (1967). Location of specificity and allotypie amino acid residues in antibody Fd fragments. Cold SpT~ng Harbor Sop. count. Biol. 32, 119-127. LAFFERTY, K. J. (1963). The interaction between virus and antibody. II. Mechanism of the reaction. Virology 21, 76-90. LINSCOTT, W. D., and LEVINSON, W. E. (1969). Complement components required for virus neutralization by early immunoglobulin antibody. Proc. Nat. Acad. Sci. U. S. 64, 52@527. MUELLER, J. H. (1931). The effect of alexin in vied-antivirus mixtures. J. ~mmunol. 26,17-23. MUSCHEL, L. H., and TOUSSAINT, A. J. (1962). Studies on the bacteriophage neutralizing activity of serums. II. Comparisons of normal and immune phage neutralizing antibodies. J. Immunol. 89, 35-40. OZAKI, Y., and TABEYI, K. (1967). Studies on the neutralization of Japanese encephahtis virus. I. Application of kinetic neutr~ization t.o the measurement of the neutralizing potency of antiserum. J. Immunol. 98, 12181223. REISFELD, R. A. (1967). Heterogeneity of rabbit light-polypeptide chains. Cold Spring Harbor Symp. Quant. Biol. 32, 291-298. SARVAS, H., and MBKE& 0. (1970). Haptenated bacteriophage in the assay of antibody quantity and aflinity: maturation of an immune response. Irnrn~~oc~~i~tr~ 7, 933-943. S~ORDINTSEV, A. A., and YABROW, A. A. (1963). Intensity of the interaction of antibody and viruses in the haemagglutination inhibition test in the presence of non-specific thermolabile stimulator of antibodies. Acta Viral. (Prague) 7, 138-151. SPRING, S. B., NISONOFF, A., and DRAY, S. (1970). Independence of allotypic determinants and antibody specificity. J. Immuno~. 105,653-660. STYK, B. (1962). Cofactor and specific antibodies against influenza viruses. III. The potentiating effect of cofactor on specific antibodies of early immune and hyperimmune sera and the differences in the character of these antibodies. Acta Virol. (Prague) 6, 327-337. SVEHAO, S.-E., ~~~BLoTH, B. (1967). Examination of virus-yM antibody complexes by electron microscopy. I. Confi~ration of -&% antibodies and mode of attachment to antigen. Virology 31, 676-687. TOWSSALNT, A. J., and MUSCHBL, L. H. (1962). Studies on the bacteriophage neutralizing activity of serums. I. An assay procedure for normal antibody and complement. J. Immtinol. 89, 27-34. KELUS,
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WALKER, W. S., LIU, C. T., and ADLER, F. L. (1969). The detection of anti-phage antibodies by passive hemagglutination. J. lmmunol. 103, 907-916. W.~LKER, W. S., FISHMAN, M., and ADLER, F. L. (1971). Detection of anti-phage antibodies by passive hemagglutination. II. Early antibodies produced in vivo and in vitro. J. Immunol. 107, 953%95ci.
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WAY, H. J., and GARWES, D. J. (1970). Serum accessory factors in the measurement of arbovirus neutralization reactions. J. Gen. Viirol. 7, 211223. YOSHINO, K., and TANIGUCHI, S. (1965). Studies on the neutralization of herpes simplex virus. I. Appearance of neutralizing antibodies having different grades of complement reqllirement. Virology 26, 44-53.