- Email: [email protected]
Serum opsonic and bactericidal activity after Neisseria meningitidis group A + C and Haemophilus influenzae type b vaccination: a comparison of three functional assays
Serodiagnosis & Immunotherapy ,N INFECTIOUS
Serodiagnosis
and Immunotherapy
in Infectious
Disease
8 (1997)
DISEASE
213-220
Serum opsonic and bactericidal activity after Neisseria meningitidis group A + C and Haemophilus injluenzae type b vaccination: a comparison of three functional assays S. Hugosson”.*, H. Fredlundb, H. Kgyhty”, P. Olcknb “Dept. bDepr.
of Clinical
of Otorhinolar~ngolog,v. Microbiology
&ehro
Medical
Center
Hospital,
S-701
85 &rho.
Sbcedm
S-701
85 &ebro,
und Immunology, &rhro Medical Crnrer Hospitul, ‘,Yutional Public Health Institure. Helsinki. Finland
Received
23 October
1996; accepted
18 November
Sweden
1996
Abstract Three functional assays on immunity, serum bactericidal activity (SBA), phagocytic killing (PK) and an index calculation of chemiluminescence induced in polymorphonuclear leukocytes (CL-index), were compared with an enzyme linked immunoassay (ELISA) of the concentration of the total Ig-fraction of anticapsular antibodies in 28 healthy adults immunized against Haemophilus influenzae type b (Hib) or JVeisseriameningitidis serogroup A and C (MC A + C). The immunization evoked a serum response measurable with all assays and a high concentration of antibodies against McA, McC and Hib usually was associated with functional activity and vice versa. However, qualitative differences were found and five of the vaccines (18%) were non-responders in functional assays despite a significant seroconversion measured by ELISA. A functional test may prove valuable in bacterial vaccine trials as complementary to immunochemical calculations of anticapsular antibodies. For estimates of opsonic activity bactericidal serum activity interferes in the PK assay. which is demonstrated in the present study. As an alternative, the CL assay, with the index technique described, is a reliable, less laborious and effective test on opsonophagocytosis. 0 1997 Elsevier Science B.V.
Keywords: H. ir$luen:ae: N. meningifidis: Vaccination;
Chemiluminescence:
1. Introduction H. infiuenzae type b (Hib) and N. meningitidis serogroups A, B and C (McA-C) can cause invasive infections such as septicemia and/or meningitis [1,2]. Hib is also the most prevalent bacteria associated with acute epiglottitis [3]. Human host-defence against invasive bacterial disease mainly comprises serum bactericidal activity and phagocytosis by polymorphonuclear leukocytes (PMNL). Both mechanisms are mediated by antibodies and complement factors [4-61. There are efficient vaccines available for protection against infec-
* Corresponding 103301.
author.
Tel.: + 46 19 15 1000; fax: + 46 19
088%0786:97:$17.00 C 1997 Elsevier PI1 SO888-0786(96)01082-7
Science
B.V.
All rights
reserved
Immunologic
tests
tions caused by McA, C, W135, Y and Hib [7,8], but so far the efforts to develop a generally accepted and efficient vaccine against McB has failed [9]. The traditional way to estimate immunity to Hib and McA and C has been immunochemical calculation of serum capsular polysaccharide (PS) antibody concentrations [lO,l 11. However, concerning McB the capsular PS is poorly immunogenic [12], and other membrane bound antigens have been suggested as important for protection. Until now there is no consensus on which McB antigens are responsible for protective immune response in humans. Assays that measure the capacity of serum factors to mediate bactericidal activity and/or phagocytosis are of interest since the functional quality of both antibodies
214
S. Hugosson
et nl.
Serodiugnosis
and Itnmunotherup~~
and complement are taken into consideration [6,13,14]. The effect measured with functional assays resemble the in vivo situation and their applicability is dependent on the questions that are addressed. A drawback with the phagocytic killing (PK) assay is that serum bactericidal action (SBA) interferes with the detection of opsonins to Hib and MC [15,16]. The measurement of light emission or chemiluminescence (CL) by leukocytes which are phagocytozing opsonized bacteria has been shown to be an alternative for evaluation of serum opsonins against both Hib and MC following infection and vaccination [17-231. The aim of the present study was to investigate the impact of vaccination, in healthy adults, against McA + C and Hib on serum opsonic and bactericidal properties. Three functional tests were compared with an enzyme linked immunoassay (ELISA) for antibody concentrations in measuring immunity against Hib and McA + C.
2. Materials
and methods
2.1. Vaccinuted indiiciduals A total of 28 voluntary adults ( > 18 years old) were vaccinated after informed consent. They were all without a history of invasive bacterial infection or compromising illness. Five of them were vaccinated by an injection of 0.5 ml meningococcal A + C polysaccharide (Institut Merieux. Lyon, France). Twelve were vaccinated with 0.5 ml Hib-PS conjugated to diphteriatoxin (PRP-D) (Prohibit”. Connaught) and 11 with 0.5 ml Hib-PS conjugated to outer membrane protein complex of McB (PRP-OMPc) (PedvaxHib”, Merck, Sharp and Dohme). All vaccines were subcutaneously administered in the deltoid region of the left arm. Blood samples were drawn before and 28 days after vaccination. Sera were immediately stored in aliquots of 1 ml at - 70°C to preserve complement activity.
in Injrctious
Disease
8 (1997)
213-220
cerebrospinal fluid or blood from patients with meningitis/septicemia. Additionally, an MC reference strain of serogroup X (NCTC 10790, Colindale, London) was used in the CL assays. The McB strain was used in the CL assays as a negative control to McA + C vaccination. A Staphylococcus epidermidis strain (L-9) was included as a control in each CL experiment as previously described [24]. All three Hi strains were cultured in Hem-l broth (Falsen, Dept. of Bacteriology, University of Gothenburg) and the three MC strains in MiillerHinton broth at 35-37°C and 5% CO,. The bacteria were cultured with mild agitation to log phase or stationary phase. After centrifugation the organisms were washed and suspended in PBS with pH adjusted to 7.4. The bacteria were counted in a Biirker chamber and concentration adjusted to 10’ colony forming units (CFU)jml. 2.3. Polymorphonuclear
leukocytes (PMNL)
Polymorphonuclear leukocytes were obtained from healthy blood donors and separated by Ficoll-Paque centrifugation (Kabi-Pharmacia, Sweden) ad modum Biiyum [25] with minor modifications as previously described [24]. More than 95% of the cells were PMNL, and trypan blue staining showed more than 95% viable cells. 2.4. Anti-capsular
polysaccharide antibodies
The concentrations of total Ig antibodies to MC serogroup A and C and Hib capsular PS were determined by ELISA as described [26-281. For meningococcal antibodies an in-house reference serum standardized in RIA with the previously used ECG reference serum [29] was used. For Hib antibodies a reference serum received from Dr. Carl Frasch (CBER, Rockville, MD) that contained 70 pg of anti-Hib PS/ ml, was used. 2.5. Chemiluminescence (CL) and index calculation
2.2. Bacteria and culture The Hib strain IH 20113 isolated from blood (National Public Health Institute, Helsinki) was used in the three functional assays. The H. injfuenzae type c (Hit) IH 20114. isolated from blood (National Public Health Institute, Helsinki), was used in the CL assays as the reference strain for the index calculation. The non-typable H. influen:tre (Hint) strain 75-00555, isolated from sputum, was used in the CL assays as a negative control to Hib vaccination. A standard panel of MC strains containing serogroups A:NT. B: 15:NT and C:NT were used for the three functional assays as described previously [24]. All three meningococcal strains were isolated from
A luminometer (LKB Wallac 1251, Turku, Finland) connected to an Olivetti Ml9 computer was used to measure luminol enhanced CL induced in PMNL. The LKB Wallac 125 1- 124 Phagocytosis program adapted for the computer was used for the measurements and calculations of data. The experimental design described before was used [24]. In brief, the following was added to each test tube (LKB Pharmacia, Uppsala Sweden No. 2174-086): 200 ~1 PBS, 100 ~1 serum (with preserved complement activity), 100 ~1 PMNL suspension (3-6 x lo6 cells/ml), 50 ~1 lo-’ M Luminol (Sigma Chemical Co., St. Louis, USA) and 50 ~1 of bacterial suspension (10’ CFU:ml PBS). The measurements were always started within 2 min after the bacterial suspen-
S. Hugossorl
~1 al.
Serodiagnosis
and Immunorlwrapy
sion had been added to the test system. The assays were performed at 37°C with continuous mixing in the measurement chamber. Each sample was measured every 194 s for at least 32 min. The S. epidrrmidis strain was included as a positive control in each experiment. The luminol-enhanced CL response induced in PMNL by MC and Hi of a particular strain opsonized with pre- and postvaccination serum was used to measure serum’s opsonizing capacity, which was given a CL index value related to the responses obtained with the reference system. The technique for the CL index calculations was used to circumvent the problems with CL response variations with different sets of PMNL and other day-to-day variations. Briefly, the MC serogroup X strain was used as the reference in the CL assays involving MC and Hi type c was the reference in Hi assays. For CL index calculation each serum’s maximum rate constant (MRC, mV/min) was divided with the MRC value induced by the reference strain with prevaccination serum. Thus, the MRC induced by the reference strains opsonized with prevaccination serum were given the CL index = 1. The range of possible values for the CL index was set to O-20 [18]. With the index calculation, the between assay variation of individual samples was < 15% for both Hi and MC. The index variation within the assay for individual samples was < 5% for both types of bacteria. 2.6. Assuys on serum buctrricidul phagocytic killing (PK)
actirity
(SBA) und
The PK was adopted from Virji and Heckels with minor modifications [30]. Bacteria were cultured to log phase or stationary phase. Bacteria (10’ CFU/ml), PMNL (106/ml) and serum were mixed. The final serum concentration was 2.5% During mild agitation bacterial survival was checked at 15, 30, 60 and 120 min by serial dilution before culture on plates. The plateau value which was reached after 30-60 min will be presented. In the SBA assay, PMNL were omitted and replaced by PBS. The killing of bacteria was measured by the ‘“log decrease of CFU. The range of possible values for SBA and PK was O&3. Throughout all the experiments with CL, SBA and PK assays the vaccines’ own serum complement were used as an internal complement source. CH,,, was checked in each individual serum to ensure that it was within normal limits. 2.7. Stutisticul unulJ.sis The statistical calculations were made using the nonparametric methods of Wilcoxon’s rank sum test for comparing one sample under two conditions and the Mann-Whitney u-test for two sample tests. A regression analysis was made with use of the correlation coefficient for a linear correlation between the assays.
in Infectious
Disease
8 (1997)
213-220
215
2.8. Et&-s The study was approved by &ebro ethical committee.
County Council’s
3. Results Immunization with the MC A + C PS evoked an antibody response measurable by ELISA in all five vaccinees with a 20-535 fold increase in anti-McA and -McC (Table 1, Fig. la-c). The CL index for McA and McC was measured for all five pairs of sera. It increased by a factor of 2-6 in all but one vaccinee who remained on the same low McC-CL index, even though he had an increase from 0.2 to 15.4 @g/ml in anti-McC measured by ELISA (subject no. 1 in Fig. la). SBA was determined for four pairs of sera (Table 1). Two of them had a 12- 13- fold increase in bactericidal antiMcC activity. The two others failed to respond with bactericidal anti-McC antibodies (no. 1 and 2 in Fig. lb), though they had an increase from 0.2 to 15.4 and 33.7 lug/ml, respectively, in anti-McC measured with ELISA. All four subjects responded with a 1.5-3.8-fold increase in SBA to McA (Fig. lb). PK was assayed for the same four pairs of sera as SBA (Table 1). All Table I The arithmetic mean and range of CL-index. total Ig fraction of anticapsular serum antibodies measured with ELISA (pg:ml) and “‘log decrease of CFU in PK and SBA in pre- (day 0) and postvaccination sera (day 28) Bacteria Vaccine
Assay
Day
CL-index ELISA
0.84 (0.6-1.1) 1.36 (0.8-3.2)
2.22 (1.3-3.4) 37.0 (22.5-63.7)
PK SBA
1.5 (0.4-2.2) 1.2 (0.3-2.0)
2.5 (1.7Y3.0) 2.3 (0.8-3.0)
CL-index ELISA
0.3 (0.1-0.5) 0.52 (0.2- 1.6)
PK SBA
0.1 (0.1-0.1) 0.1 (0.1-0.1)
1.2 (0.3-1.6) 57.9 (15.4107.1) 1.9 (0.9-2.6) 0.8 (0.1-1.3)
CL-index PK
0.7 (0.4- I .O) 0.3 (0.2-0.5)
0.6 (0.3-I .O) 0.3 (0.2-0.5)
SBA
0.3 (0.1-0.5)
0.3 (0.2-0.4)
Hib PRP-OMPc (n= II)
CL-index ELISA PK SBA
0.9 2.8 1.I 0.9
(0.4- I .9) (0.7-7.1) (0.2-2.8) (0.1-2.2)
1.6 27.2 2.2 1.9
Hib PRP-D (n = 12)
CL-index ELlSA PK SBA
1 .o 3.7 1.2 1.0
(0.4- 1.4) (0.7- 17. I) (0.3-2.7) (0.03-2.5)
4.9 (1.7-9.2) 174.4 (32.6-700) 2.2 (I .6&2.6) 2.1 (1.4-2.7)
McA MC A+C tine (II = 5) McC MC A+C tine (I? = 5) McB MC AfC tine (n = 5)
vac-
vac-
vat-
0
Day
28
(0.8-4.6) (4.3-86.6) (1.3-2.8) (1.0-2.8)
216
S. Hugosson
et ul.
Serodiagnosis
and Immunotherap?:
Fig. I. The response to McA + C vaccination (n = 5). Functional assays compared to total Ig fraction of anti-McA PS (ELISA) on day 0 (3) and 28 (0) and of anti-McC PS on day 0 (A) and 28 (A) postvaccination. The numbers 1 and 2 correspond to individuals commented on in the text. (a) CL-index vs. ELISA; (b) SBA vs. ELISA; (c) PK vs. ELISA.
in Infectious
Disease
8 (1997)
213-220
subjects had an increased activity to both McA and C by a factor of 1.5-25 (Fig. lc). Vaccination against McA and C did not affect the CL-index, SBA or PK for McB (Table 1). Eleven of 12 individuals vaccinated with PRP-D responded by a 20-220-fold increase in serum concentrations of anti-Hib PS (Table 1, Fig. 2a-c). One male (no. 3 in Fig. 2a-c), who had the highest prevaccination concentration, responded by a factor of 2.7 (to 46.1 pg/ml). In the CL assay 11 subjects (the same as above) had a 2-14-fold increase in the CL-index. The deviant subject had a 1.7-fold increase (no. 3 in Fig. 2a). Nine of the 12 subjects responded by a factor of 1.5-74 in SBA. Two subjects responded by a factor 1.1~ 1.4 and one individual failed to increase bactericidal activity. This man (no. 4 in Fig. 2b) had an increase in serum concentration of anti-Hib PS from 4.7 to 109.3 lug/ml (ELISA). In the PK assay 9 subjects responded by a factor of 1.5 to 75. One subject slightly (1.3-fold) increased PK and 2 persons (no. 3 and 4 in Fig. 2c) failed to respond. Four of 1 I individuals vaccinated with PRP-OMPc responded by a 20-27-fold increase in serum anti-Hib PS measured with ELISA (Table 1, Fig. 2d-f). Two had a lo- 15-fold increase; 3 had a 1.5-9-fold increase and 2 subjects (no. 5 and 6 in Fig. 2d-f) failed to respond. In the CL assays the index was raised 2-3.8fold in 4 subjects namely from those with the > 20-fold Ig-response. Four subjects had an index rise by a factor 1.5-1.9 and 3 did not respond (no. 5-7 in Fig. 2d). Eight subjects evoked a response by a factor of 1.5-10 in SBA and PK whereas 3 individuals failed to respond in these assays (no. 5, 6 and 8 in Fig. 2e and fl. The five vaccinees, no. 1, 2, 4, 7 and 8, did not respond in one or several functional assays, though they significantly raised their serum antibody concentration, which makes a non-response frequency of 18%. There was a significantly lower CL-index and antibody (total Ig) concentration measured with ELISA (P < 0.001) in the group receiving PRP-OMPc compared to PRP-D in postvaccination sera. However, no significant difference between the two vaccines was found in the SBA and PK assays (Table 1 and Fig. 2a-f). In the postvaccination phase the mean response of bacterial killing was higher with PK than with SBA for McA + C and Hib but the difference was not significant (P > 0.05) for any of the bacteria (Table 1). With use of regression analysis the correlation between the assays were calculated and the coefficients for a linear correlation are presented in Table 2. The CL-index (arithmetic mean and range, n = 23) induced by Hib before vaccination was 0.9 (0.4- 1.9) and for Hint 1.9 (0.7-3.9), P < 0.01. Vaccination against Hib did not affect the CL response induced by the Hint: CL-index at day 28 being 2.0 (0.6-4.4).
S. Hugo~son
Fig. 2. The of anti-Hib d) CL-index
et ul. , Scwdiagnosis
und Inmunotlwapy
in Infec‘rious
Disease
8 (1997)
213-220
response to vaccination uith PRP-D (n = 12; A-C) or PRP-OMPc (n = I I; D-F). Functional assays compared PS (ELISA) on day 0 ( _ ) and 28 (+) postvaccination. The numbers 3-8 correspond to individuals commented vs. ELISA; (b. e) SBA vs. ELISA: (c. f) PK vs. ELISA.
217
to total Ig fraction on in the text. (a,
218
S. Hugosson
et al.
Srrodiagnosis
and
Immunotherup~~
The CL responses induced by the indexing McX- and Hit-reference strains were mainly unchanged ( < 10% variation) with use of pre- and postvaccination sera. In the CL assays PMNL alone as well as PMNL in the presence of unopsonized bacteria only gave background light emission.
4. Discussion The most relevant way of estimating protection against infection in a population following immunization with a bacterial vaccine is a clinical field trial. However, a field trial necessitates considerable amounts of time and resources. Reliable in vitro methods for estimating individual protection are therefore desirable although they cannot encounter the priming effect on the immune system induced by a vaccination. ELISA measures all antibodies directed to a particular antigen whereas their functional activity remains unknown. The concentration of serum antibodies that is enough to give protection against MC infection is not known exactly [16]. The protection level of anti-PS antibodies against Hib-infection has been suggested to lie between 0.1 and 1.O pg/ml [3 11. However, functional assays have shown a striking heterogenicity in functional activity of the Ig fraction following immunization against Hib-infection [ 14,32.33]. The variability has been related to differences in antibody specificity and avidity between and within Ig classes. For evaluation of the proportion of non-functional antibodies it is desirable to use functional assays such as complement-mediated bactericidal activity or opsonic activity in bacterial vaccine trials [14,34,34]. In these assays a human complement source is desirable since complement from other species is not as reliable [16]. The present investigation, of the opsonic and bactericidal activity induced by McA + C and Hib vaccination, in voluntary adults may be regarded as a pilot study. It was designed to compare and correlate the results of the functional assays with the concentration of serum antibodies measured by ELISA. Of special interest was to compare the results of serum opsonic Table 2 Coefficients
for
a linear
correlation
between
the
results
of the three
functional assays (CL-index, SBA and PK) and the total Ig fraction of anti-capsular serum antibodies against MC A C and Hlb measured with ELISA before vaccination (bold) and the coefficients for linear correlation tion sera
ELISA CL-index SBA
between (italics)
the fold
increase
of serum
CL-index
SBA
0.40
0.47 0.51
0.45
activity
in postvaccina-
PK 0.04 0.15
0.54 0.49
0.04 0.18
0.96 0.98
in Infrcrious
Disease
8 (1997)
213-220
activity measured by CL index and PK. The CL assay estimates the opsonizing activity of antibodies. The internal complement activity of the test sample is preserved in each experiment. The oxidative burst that is necessary to induce CL is dependent on the bacteriaPMNL interaction in the presence of opsonizing serum antibodies and complement. Thus. the bactericidal activity is not taken into account with the CL index technique. It is an indirect measurement of opsonophagocytosis without being in need of radioactive agents. The index calculation with the index- and control strains minimizes the risk of intra- and interassay variations though non-relevant PMNL-bacteria interaction may influence the results. The SBA and PK assays measure antibody-mediated bactericidal activity with preserved internal complement activity. By definition PK also takes the additional effect of opsonophagocytosis into consideration. They are robust and direct methods of bacterial killing but have the drawback of being laborious and time consuming. With the limitations of internal complement to dilute enough. the functional methods cannot discriminate bacterial killing between individuals with high antibody concentrations. This drawback reduces the validity of the tests, and this is more obvious with SBA and PK than with the CL-index technique. In the present study. the ELISA and the CL assay defined PRP-D as a better immunogen than PRPOMPc. The results presented in Fig. 2 also indicate that PRP-OMPc induces a less CL-index activity per pg anti-HibPS (ELISA) than PRP-D while the SBA and PK did not detect this qualitative difference. This may be due to a population of antibodies of lower avidity induced by PRP-OMPc, which is detected as reduced opsonic activity in the CL assay. The bactericidal activity of vaccine-induced antibodies, measured by SBA and PK. was the same for the two vaccines. This may suggest that there is no inter-vaccine difference of functional antibody properties such as avidity in conferring bactericidi, but it can also be a result of the inability of these assays to discriminate between the high serum activity that follows from immunization of adults by a conjugated Hib-vaccine. Furthermore, from the results of the PK assay there is no obvious way of estimating the proportion of opsonic activity because of the bactericidal interference. SBA is not as complex as PK and a bactericidal assay is most often adopted to detect functional activity of antibodies [6,14]. Qualitative differences in SBA, in contrast to the ELISA antibody results, were found in a recent trial of two MC A + C vaccines [36]. In McB vaccine trials there are reports of both poor [6] and good correlation [35] between serum concentrations of antibodies against various surface antigens of McB and serum bactericidal activity.
S. Hugosson
et al. : Serodtagnosis
and
Immunotl~erap~
In our study the mean response was good by all assays indicating that the generated antibodies acted functionally in most individuals. Interindividual differences in response characteristics existed but usually high antibody concentrations were associated with functional activity and vice versa. A possible explanation to poor functional activity despite high concentrations of antibodies is the prevalence of blocking antibodies, antibodies of low avidity or of non-functional Ig classes [14]. It has been suggested that IgA can inhibit the interaction between IgG and the bacteria which bind complement and mediate cell lysis or phagocytosis [32,37]. A proportionally high antibody response of IgA class to vaccination could be non-physiological in this respect. In our study one of the vaccines, who responded well in the ELISA but poor in the functional assays, had an exceptionally high IgA concentration in postvaccination serum (data not shown). The other non-responders had the equally high proportion of IgG/IgA ratio as the responders. The serum response measured with ELISA correlated better with CL than with the other two functional assays. This could imply that a main fraction of the antibodies generated by vaccination acted as opsonins. We found that the bacterial killing by PK in the presence of PMNL is somewhat but not significantly higher than in SBA suggesting that the activity measured with PK mainly is of bactericidal origin and the opsonizing properties induced by the vaccination probably are poorly considered. This suggestion is supported by the very strong correlation between the SBA and PK assays shown in Table 2. The advantage of Hib to Hint in resisting phagocytosis is shown in our CL experiment with prevaccination sera. In accordance with the specificity of antibodies induced by immunization, the CL activity against Hint and McB was unaffected in postvaccination sera following vaccination against Hib PS/McA + C, and these experiments can serve as negative controls. In future vaccine trials, an immunochemical calculation (ELISA or RIA) of serum concentrations of antibodies against relevant bacterial antigens is mandatory. With regard to the existence of antibodies that are reduced in specificity, of low avidity or of non-functional Ig class a functional assay can be complementary to make sure that an antibody response correlates with bactericidal or opsonic activity or passive protection in an animal model [23,34,36]. Functional assays are logical to use in trials with cellular vaccines such as McB outer membrane preparations, where the important antigens for protection are unknown. For measurements of serum opsonic activity the results of PK may be confusing because of the strong influence by the bactericidal activity. The CL index technique estimates this property in a more unaffected way. To evaluate the CL assay further it would be desirable to compare it
in lnjectious
Disease
8 (1997)
213-220
219
with other in vitro assays of opsonophagocytosis such as uptake of radioactive bacteria by PMNL and flow cytometry. It would also be of interest to correlate it with an animal model of passive protection.
Acknowledgements We wish to thank the Research Committee County Council for financial support.
of Grebro
References [II
Berg S, Trollfors B, Alestig K, Jodal U. Incidence, serogroups and case-fatality rate of invasive meningococcal infections in a Swedish region 197551989. Stand J Infect Dis 1992: 24: 333338. of PI Make12 PH, Takala AK. Peltola H, Eskola J. Epidemiology invasive Haemophilus injuenzae type b disease. J Infect Dis 1992; 165 (Suppl I): 2-6. S, Olcen P, Ekedahl C. Acute epiglottitis-etiology, [31 Hugosson epidemiology and outcome in a population before large-scale Haemophilus influenrae type b vaccination. Clin Otolaryngol 1994; 19: 44-5. MM, Christoffer NC. Griffiss JM, Baker CJ, Man141 Estabrook drel] RE. Sialylation and human neutrophil killing of group C Neissrria meningitidis. J Infect Dis 1992; 166: 1079988. basis of invasive H. irzjluenrae type b [51 Moxon ER. Molecular disease. J Infect Dis 1992; 165 (Suppl): 77781. [61 Milagres LG, Ramos RS, Sacchi CT. Melles CEA, Vieira VSD, Sato H. et al. Immune response of Brazilian children to Neisseria meningitidk serogroup B outer membrane protein vaccine: comparison of efficacy. J Infect Dis 1994; 62: 4419-24. A. Meningococcal disease and its control with meningo[71 Galazka coccal polysaccharide vaccines, Bull WHO 1982; 60: l-7. PI Murphy TV. White KE, Pastor P, Gabriel L, Medley F, Granoff DM. et al. Declining incidence of Haemophilus influenzar type b disease since introduction of vaccination. J Am Med Assoc 1993: 269: 24668. JK. Arnesen 0, Fredriksen JH, [91 Bjune G, Hoiby EA, Gronnesby Halstensen A. Effect of outer membrane vesicle vaccine against group B meningococcal disease in Norway). Lancet 1991; 338: 109336. DK, Baker CJ. 1101Griffiss J, Brandt BL, Broud DD, Gorofff Immune response of infants and children to disseminated infections with Neisseria meningitidis. J Infect Dis 1984; 150: 71-9. H, Hakulinen M. [‘II Eskola J. Peltola H. Takala AK, Kayhty Karanko V, et al. Efficacy of H. in@wtzar type b polysaccharide-diphtheria toxoid conjugate vaccine in infancy. N Engl J Med 1987; 317: 717-22. of complement source [I21 Zollinger WD. Mandrel] RE. Importance in bactericidal activity of human antibody and murine monoclonal antibody to meningococcal group B polysaccharide. Infect Immun 1983; 40: 257764. I. Gotschlich EC, Artenstein MS. Human immu1131Goldschneider nity to the meningococcus. I. The role of humoral antibodies. J Exp Med 1969: 129: 1307726. DM. Variability in the functional ['41 Amir J, Liang X, Granoff activity of vaccine-induced antibody to Harmophilus inJlurn:ae type b. Pediatr Res 1990; 27: 358-64. SL. Waldo B, Johnston RB. Separation of serum [I51 Newman bactericidal and opsonizing activities of Harmop/~ilus injuenzae type b. Infect lmmun 1973; 8: 164-6.
220 [I61
[17]
(IS]
[19]
[20]
[21]
[22]
[23]
[24]
[25] [26]
[27]
S. Hugosson
et a/.
Serodiagttosb
and Immurrofherap~
Fredlund H. Serum factors and polymorphonuclear leukocytes in human host defence against Neissrria mrningitidis. Studies of interactions with special reference to a chemiluminometric technique. Stand J Infect Dis 1993: SX7: l-72. Kaplan SL. Umstead CL. Mason EO. Anderson DC. Parke Jr JC, Feigin RD. Assessment of Huemophilus mfluenzar type b opsonins by neutrophil chemiluminescence. J Clin Microbial 1981: 13: 532-9. Fredlund H, Sellden H, Danielssom D, Olcen P. Early development in healthy children of serum opsonins against nonpathogenie Nrissrricr meningitidis. APM IS 1992; 100: 449- 54. Lehmann V, Solberg CO. Group B meningococci opsonins in serum measured by polymorphonuclear leukocyte chemiluminescence. APMIS 1980; X8: 227731. Guttormsen HK. Bjerknes R. Halstensen A. Naess A. H&by A. Solberg CO. Cross-reacting serum opsonins to meningococci after vaccination. J Infect Dis 1993; 167: 1314.-9. Halstensen A, Haneberg B. Froholm 0. Lehmann V, Frasch CE, Solberg CO. Human opsonins to meningococci after vaccination. Infect Immun 1984: 46: 67336. Crook G. Wilson R, Kroll S. Todd H. Garbett N. Moxon R. et al. Opsonic requirements and interaction of Harmophilus injluflltm:rre with human polymorphonuclear neutrophil leucocytes studied by luminol-enhanced chemiluminescence. Microb Pathog 19x9; 7: 101-10. Ojo-Amaize EA. Church JA. Barka NE, Agopian MS, Peter JB. A rapid and sensitive chemiluminescence assay for evaluation of functional opsonic activity of Htremophrlus influenxw type b specific antibodies. Clin Diagn Lab Immunol 1995; 2: 2X6-90. Fredlund H, Olcen P. Danielsson D. A reference procedure to study chemiluminescence induced in polymorphonuclear leukocytes by :Veisseria /rrrnirrgirit/i.c. APMIS 1988: 96: 941 -9. Boyum A. Separation of blood leukocytes, granulocytes and lymphocytes. Tissue Antigens 1974: 4: 269974. Arakere G. Frasch CE. Specificity of antibodies to O-acetyl-positive and 0-acetyl-negative group C meningococcal polysaccharides in sera from vaccinees and carriers. Infect Immun 1991; 59: 4349.. 56. Platonov AE, Beloborodov VB, Pavlota LI. Vershinina IV. Kayhty H. Vaccination of patients dehcient in a late complement
in Irtfectious
Disease
8 (1997)
213-220
component with tetravalent meningococcal capsular polysaccharide vaccine. Clin Exp Immunol 1995; 100: 32-9. S. Klyhty H. Saarinen L. Ronnberg P-R, Eskola J. [2X1Kurikka Immunity after one dose of Haemophilus in&n;ae type b conjugate vaccine. J Infect Dis 1995; 172: 1268-72. EC, Rey M. Triau R, Sparks KJ. Quantitative deter~291 Gotschlich minations of human immune response to immunization with meningococcal vaccines. J Clin Invest 1972; 51: 89996. JE. The effect of protein II and pili on the [301 Virji M, Heckels interaction of Nrisseria gonorrhoea with human polymorphonuclear leukocytes. J Gen Microbial 1986; 132: 503-12. DM, Holmes SJ. Comparative immunogenicity of [311 Granoff Haemophilu.c in/luen:ar type b polysaccharide-protein conjugate vaccines. Vaccine 1991; 9 (Suppl): 530.-4. JR, Barrus V, Cates KL, Siber CR. Functional charac[321 Schreiber terization of human IgG, IgM and IgA antibody directed to the capsule of Haemophilus iny%en;ae type b. J Infect Dis 1986; 153: 8-15. [33] Lucas AH, Granoff DM. Functional differences in idiotypically delined IgGl anti-polysaccharide antibodies elicited by vaccination with Haemopldus injurnzae type b polysaccharide protein conjugates. J Immunol 1995; 154: 41955202. DM. Lucas AH. Laboratory correlates of protection [341 Granoff against Haw~opldus in/?uen:ue type b disease. Importance of assessment of antibody avidity and immunologic memory. Ann NY Acad Sci 1995: 754: 278X88. A, Rosenqvist E, Pedersen AK, [351 Aase A, Bjune G. Hoiby Michaelsen T. Comparison among opsonic activity, antimeningococcal immunoglobulin G response and serum bactericidal activity against meningococci in sera from vaccinees after immunization with a serogroup B outer membrane vesicle vaccine. Infect Immun 1995: 63: 3531-6. JM, Chiu SS, Wong VK, Partridge S, Chang S, Chiu [361 Lieberman C et al. Safety and immunogenicity of a serogroups A/C Neisseriu mmingitidis oligosaccharide-protein conjugate vaccine in young children. J Am Med Assoc 1996; 275: 1499-503. J. Epidemic meningococcal disease: a synthesis of a [371 Grifliss hypothetical immunoepidemiologic model. Rev Infect Dis 1982; 4: 159 71.
Serodiagnosis
and Immunotherapy
in Infectious
Disease
8 (1997)
DISEASE
213-220
Serum opsonic and bactericidal activity after Neisseria meningitidis group A + C and Haemophilus injluenzae type b vaccination: a comparison of three functional assays S. Hugosson”.*, H. Fredlundb, H. Kgyhty”, P. Olcknb “Dept. bDepr.
of Clinical
of Otorhinolar~ngolog,v. Microbiology
&ehro
Medical
Center
Hospital,
S-701
85 &rho.
Sbcedm
S-701
85 &ebro,
und Immunology, &rhro Medical Crnrer Hospitul, ‘,Yutional Public Health Institure. Helsinki. Finland
Received
23 October
1996; accepted
18 November
Sweden
1996
Abstract Three functional assays on immunity, serum bactericidal activity (SBA), phagocytic killing (PK) and an index calculation of chemiluminescence induced in polymorphonuclear leukocytes (CL-index), were compared with an enzyme linked immunoassay (ELISA) of the concentration of the total Ig-fraction of anticapsular antibodies in 28 healthy adults immunized against Haemophilus influenzae type b (Hib) or JVeisseriameningitidis serogroup A and C (MC A + C). The immunization evoked a serum response measurable with all assays and a high concentration of antibodies against McA, McC and Hib usually was associated with functional activity and vice versa. However, qualitative differences were found and five of the vaccines (18%) were non-responders in functional assays despite a significant seroconversion measured by ELISA. A functional test may prove valuable in bacterial vaccine trials as complementary to immunochemical calculations of anticapsular antibodies. For estimates of opsonic activity bactericidal serum activity interferes in the PK assay. which is demonstrated in the present study. As an alternative, the CL assay, with the index technique described, is a reliable, less laborious and effective test on opsonophagocytosis. 0 1997 Elsevier Science B.V.
Keywords: H. ir$luen:ae: N. meningifidis: Vaccination;
Chemiluminescence:
1. Introduction H. infiuenzae type b (Hib) and N. meningitidis serogroups A, B and C (McA-C) can cause invasive infections such as septicemia and/or meningitis [1,2]. Hib is also the most prevalent bacteria associated with acute epiglottitis [3]. Human host-defence against invasive bacterial disease mainly comprises serum bactericidal activity and phagocytosis by polymorphonuclear leukocytes (PMNL). Both mechanisms are mediated by antibodies and complement factors [4-61. There are efficient vaccines available for protection against infec-
* Corresponding 103301.
author.
Tel.: + 46 19 15 1000; fax: + 46 19
088%0786:97:$17.00 C 1997 Elsevier PI1 SO888-0786(96)01082-7
Science
B.V.
All rights
reserved
Immunologic
tests
tions caused by McA, C, W135, Y and Hib [7,8], but so far the efforts to develop a generally accepted and efficient vaccine against McB has failed [9]. The traditional way to estimate immunity to Hib and McA and C has been immunochemical calculation of serum capsular polysaccharide (PS) antibody concentrations [lO,l 11. However, concerning McB the capsular PS is poorly immunogenic [12], and other membrane bound antigens have been suggested as important for protection. Until now there is no consensus on which McB antigens are responsible for protective immune response in humans. Assays that measure the capacity of serum factors to mediate bactericidal activity and/or phagocytosis are of interest since the functional quality of both antibodies
214
S. Hugosson
et nl.
Serodiugnosis
and Itnmunotherup~~
and complement are taken into consideration [6,13,14]. The effect measured with functional assays resemble the in vivo situation and their applicability is dependent on the questions that are addressed. A drawback with the phagocytic killing (PK) assay is that serum bactericidal action (SBA) interferes with the detection of opsonins to Hib and MC [15,16]. The measurement of light emission or chemiluminescence (CL) by leukocytes which are phagocytozing opsonized bacteria has been shown to be an alternative for evaluation of serum opsonins against both Hib and MC following infection and vaccination [17-231. The aim of the present study was to investigate the impact of vaccination, in healthy adults, against McA + C and Hib on serum opsonic and bactericidal properties. Three functional tests were compared with an enzyme linked immunoassay (ELISA) for antibody concentrations in measuring immunity against Hib and McA + C.
2. Materials
and methods
2.1. Vaccinuted indiiciduals A total of 28 voluntary adults ( > 18 years old) were vaccinated after informed consent. They were all without a history of invasive bacterial infection or compromising illness. Five of them were vaccinated by an injection of 0.5 ml meningococcal A + C polysaccharide (Institut Merieux. Lyon, France). Twelve were vaccinated with 0.5 ml Hib-PS conjugated to diphteriatoxin (PRP-D) (Prohibit”. Connaught) and 11 with 0.5 ml Hib-PS conjugated to outer membrane protein complex of McB (PRP-OMPc) (PedvaxHib”, Merck, Sharp and Dohme). All vaccines were subcutaneously administered in the deltoid region of the left arm. Blood samples were drawn before and 28 days after vaccination. Sera were immediately stored in aliquots of 1 ml at - 70°C to preserve complement activity.
in Injrctious
Disease
8 (1997)
213-220
cerebrospinal fluid or blood from patients with meningitis/septicemia. Additionally, an MC reference strain of serogroup X (NCTC 10790, Colindale, London) was used in the CL assays. The McB strain was used in the CL assays as a negative control to McA + C vaccination. A Staphylococcus epidermidis strain (L-9) was included as a control in each CL experiment as previously described [24]. All three Hi strains were cultured in Hem-l broth (Falsen, Dept. of Bacteriology, University of Gothenburg) and the three MC strains in MiillerHinton broth at 35-37°C and 5% CO,. The bacteria were cultured with mild agitation to log phase or stationary phase. After centrifugation the organisms were washed and suspended in PBS with pH adjusted to 7.4. The bacteria were counted in a Biirker chamber and concentration adjusted to 10’ colony forming units (CFU)jml. 2.3. Polymorphonuclear
leukocytes (PMNL)
Polymorphonuclear leukocytes were obtained from healthy blood donors and separated by Ficoll-Paque centrifugation (Kabi-Pharmacia, Sweden) ad modum Biiyum [25] with minor modifications as previously described [24]. More than 95% of the cells were PMNL, and trypan blue staining showed more than 95% viable cells. 2.4. Anti-capsular
polysaccharide antibodies
The concentrations of total Ig antibodies to MC serogroup A and C and Hib capsular PS were determined by ELISA as described [26-281. For meningococcal antibodies an in-house reference serum standardized in RIA with the previously used ECG reference serum [29] was used. For Hib antibodies a reference serum received from Dr. Carl Frasch (CBER, Rockville, MD) that contained 70 pg of anti-Hib PS/ ml, was used. 2.5. Chemiluminescence (CL) and index calculation
2.2. Bacteria and culture The Hib strain IH 20113 isolated from blood (National Public Health Institute, Helsinki) was used in the three functional assays. The H. injfuenzae type c (Hit) IH 20114. isolated from blood (National Public Health Institute, Helsinki), was used in the CL assays as the reference strain for the index calculation. The non-typable H. influen:tre (Hint) strain 75-00555, isolated from sputum, was used in the CL assays as a negative control to Hib vaccination. A standard panel of MC strains containing serogroups A:NT. B: 15:NT and C:NT were used for the three functional assays as described previously [24]. All three meningococcal strains were isolated from
A luminometer (LKB Wallac 1251, Turku, Finland) connected to an Olivetti Ml9 computer was used to measure luminol enhanced CL induced in PMNL. The LKB Wallac 125 1- 124 Phagocytosis program adapted for the computer was used for the measurements and calculations of data. The experimental design described before was used [24]. In brief, the following was added to each test tube (LKB Pharmacia, Uppsala Sweden No. 2174-086): 200 ~1 PBS, 100 ~1 serum (with preserved complement activity), 100 ~1 PMNL suspension (3-6 x lo6 cells/ml), 50 ~1 lo-’ M Luminol (Sigma Chemical Co., St. Louis, USA) and 50 ~1 of bacterial suspension (10’ CFU:ml PBS). The measurements were always started within 2 min after the bacterial suspen-
S. Hugossorl
~1 al.
Serodiagnosis
and Immunorlwrapy
sion had been added to the test system. The assays were performed at 37°C with continuous mixing in the measurement chamber. Each sample was measured every 194 s for at least 32 min. The S. epidrrmidis strain was included as a positive control in each experiment. The luminol-enhanced CL response induced in PMNL by MC and Hi of a particular strain opsonized with pre- and postvaccination serum was used to measure serum’s opsonizing capacity, which was given a CL index value related to the responses obtained with the reference system. The technique for the CL index calculations was used to circumvent the problems with CL response variations with different sets of PMNL and other day-to-day variations. Briefly, the MC serogroup X strain was used as the reference in the CL assays involving MC and Hi type c was the reference in Hi assays. For CL index calculation each serum’s maximum rate constant (MRC, mV/min) was divided with the MRC value induced by the reference strain with prevaccination serum. Thus, the MRC induced by the reference strains opsonized with prevaccination serum were given the CL index = 1. The range of possible values for the CL index was set to O-20 [18]. With the index calculation, the between assay variation of individual samples was < 15% for both Hi and MC. The index variation within the assay for individual samples was < 5% for both types of bacteria. 2.6. Assuys on serum buctrricidul phagocytic killing (PK)
actirity
(SBA) und
The PK was adopted from Virji and Heckels with minor modifications [30]. Bacteria were cultured to log phase or stationary phase. Bacteria (10’ CFU/ml), PMNL (106/ml) and serum were mixed. The final serum concentration was 2.5% During mild agitation bacterial survival was checked at 15, 30, 60 and 120 min by serial dilution before culture on plates. The plateau value which was reached after 30-60 min will be presented. In the SBA assay, PMNL were omitted and replaced by PBS. The killing of bacteria was measured by the ‘“log decrease of CFU. The range of possible values for SBA and PK was O&3. Throughout all the experiments with CL, SBA and PK assays the vaccines’ own serum complement were used as an internal complement source. CH,,, was checked in each individual serum to ensure that it was within normal limits. 2.7. Stutisticul unulJ.sis The statistical calculations were made using the nonparametric methods of Wilcoxon’s rank sum test for comparing one sample under two conditions and the Mann-Whitney u-test for two sample tests. A regression analysis was made with use of the correlation coefficient for a linear correlation between the assays.
in Infectious
Disease
8 (1997)
213-220
215
2.8. Et&-s The study was approved by &ebro ethical committee.
County Council’s
3. Results Immunization with the MC A + C PS evoked an antibody response measurable by ELISA in all five vaccinees with a 20-535 fold increase in anti-McA and -McC (Table 1, Fig. la-c). The CL index for McA and McC was measured for all five pairs of sera. It increased by a factor of 2-6 in all but one vaccinee who remained on the same low McC-CL index, even though he had an increase from 0.2 to 15.4 @g/ml in anti-McC measured by ELISA (subject no. 1 in Fig. la). SBA was determined for four pairs of sera (Table 1). Two of them had a 12- 13- fold increase in bactericidal antiMcC activity. The two others failed to respond with bactericidal anti-McC antibodies (no. 1 and 2 in Fig. lb), though they had an increase from 0.2 to 15.4 and 33.7 lug/ml, respectively, in anti-McC measured with ELISA. All four subjects responded with a 1.5-3.8-fold increase in SBA to McA (Fig. lb). PK was assayed for the same four pairs of sera as SBA (Table 1). All Table I The arithmetic mean and range of CL-index. total Ig fraction of anticapsular serum antibodies measured with ELISA (pg:ml) and “‘log decrease of CFU in PK and SBA in pre- (day 0) and postvaccination sera (day 28) Bacteria Vaccine
Assay
Day
CL-index ELISA
0.84 (0.6-1.1) 1.36 (0.8-3.2)
2.22 (1.3-3.4) 37.0 (22.5-63.7)
PK SBA
1.5 (0.4-2.2) 1.2 (0.3-2.0)
2.5 (1.7Y3.0) 2.3 (0.8-3.0)
CL-index ELISA
0.3 (0.1-0.5) 0.52 (0.2- 1.6)
PK SBA
0.1 (0.1-0.1) 0.1 (0.1-0.1)
1.2 (0.3-1.6) 57.9 (15.4107.1) 1.9 (0.9-2.6) 0.8 (0.1-1.3)
CL-index PK
0.7 (0.4- I .O) 0.3 (0.2-0.5)
0.6 (0.3-I .O) 0.3 (0.2-0.5)
SBA
0.3 (0.1-0.5)
0.3 (0.2-0.4)
Hib PRP-OMPc (n= II)
CL-index ELISA PK SBA
0.9 2.8 1.I 0.9
(0.4- I .9) (0.7-7.1) (0.2-2.8) (0.1-2.2)
1.6 27.2 2.2 1.9
Hib PRP-D (n = 12)
CL-index ELlSA PK SBA
1 .o 3.7 1.2 1.0
(0.4- 1.4) (0.7- 17. I) (0.3-2.7) (0.03-2.5)
4.9 (1.7-9.2) 174.4 (32.6-700) 2.2 (I .6&2.6) 2.1 (1.4-2.7)
McA MC A+C tine (II = 5) McC MC A+C tine (I? = 5) McB MC AfC tine (n = 5)
vac-
vac-
vat-
0
Day
28
(0.8-4.6) (4.3-86.6) (1.3-2.8) (1.0-2.8)
216
S. Hugosson
et ul.
Serodiagnosis
and Immunotherap?:
Fig. I. The response to McA + C vaccination (n = 5). Functional assays compared to total Ig fraction of anti-McA PS (ELISA) on day 0 (3) and 28 (0) and of anti-McC PS on day 0 (A) and 28 (A) postvaccination. The numbers 1 and 2 correspond to individuals commented on in the text. (a) CL-index vs. ELISA; (b) SBA vs. ELISA; (c) PK vs. ELISA.
in Infectious
Disease
8 (1997)
213-220
subjects had an increased activity to both McA and C by a factor of 1.5-25 (Fig. lc). Vaccination against McA and C did not affect the CL-index, SBA or PK for McB (Table 1). Eleven of 12 individuals vaccinated with PRP-D responded by a 20-220-fold increase in serum concentrations of anti-Hib PS (Table 1, Fig. 2a-c). One male (no. 3 in Fig. 2a-c), who had the highest prevaccination concentration, responded by a factor of 2.7 (to 46.1 pg/ml). In the CL assay 11 subjects (the same as above) had a 2-14-fold increase in the CL-index. The deviant subject had a 1.7-fold increase (no. 3 in Fig. 2a). Nine of the 12 subjects responded by a factor of 1.5-74 in SBA. Two subjects responded by a factor 1.1~ 1.4 and one individual failed to increase bactericidal activity. This man (no. 4 in Fig. 2b) had an increase in serum concentration of anti-Hib PS from 4.7 to 109.3 lug/ml (ELISA). In the PK assay 9 subjects responded by a factor of 1.5 to 75. One subject slightly (1.3-fold) increased PK and 2 persons (no. 3 and 4 in Fig. 2c) failed to respond. Four of 1 I individuals vaccinated with PRP-OMPc responded by a 20-27-fold increase in serum anti-Hib PS measured with ELISA (Table 1, Fig. 2d-f). Two had a lo- 15-fold increase; 3 had a 1.5-9-fold increase and 2 subjects (no. 5 and 6 in Fig. 2d-f) failed to respond. In the CL assays the index was raised 2-3.8fold in 4 subjects namely from those with the > 20-fold Ig-response. Four subjects had an index rise by a factor 1.5-1.9 and 3 did not respond (no. 5-7 in Fig. 2d). Eight subjects evoked a response by a factor of 1.5-10 in SBA and PK whereas 3 individuals failed to respond in these assays (no. 5, 6 and 8 in Fig. 2e and fl. The five vaccinees, no. 1, 2, 4, 7 and 8, did not respond in one or several functional assays, though they significantly raised their serum antibody concentration, which makes a non-response frequency of 18%. There was a significantly lower CL-index and antibody (total Ig) concentration measured with ELISA (P < 0.001) in the group receiving PRP-OMPc compared to PRP-D in postvaccination sera. However, no significant difference between the two vaccines was found in the SBA and PK assays (Table 1 and Fig. 2a-f). In the postvaccination phase the mean response of bacterial killing was higher with PK than with SBA for McA + C and Hib but the difference was not significant (P > 0.05) for any of the bacteria (Table 1). With use of regression analysis the correlation between the assays were calculated and the coefficients for a linear correlation are presented in Table 2. The CL-index (arithmetic mean and range, n = 23) induced by Hib before vaccination was 0.9 (0.4- 1.9) and for Hint 1.9 (0.7-3.9), P < 0.01. Vaccination against Hib did not affect the CL response induced by the Hint: CL-index at day 28 being 2.0 (0.6-4.4).
S. Hugo~son
Fig. 2. The of anti-Hib d) CL-index
et ul. , Scwdiagnosis
und Inmunotlwapy
in Infec‘rious
Disease
8 (1997)
213-220
response to vaccination uith PRP-D (n = 12; A-C) or PRP-OMPc (n = I I; D-F). Functional assays compared PS (ELISA) on day 0 ( _ ) and 28 (+) postvaccination. The numbers 3-8 correspond to individuals commented vs. ELISA; (b. e) SBA vs. ELISA: (c. f) PK vs. ELISA.
217
to total Ig fraction on in the text. (a,
218
S. Hugosson
et al.
Srrodiagnosis
and
Immunotherup~~
The CL responses induced by the indexing McX- and Hit-reference strains were mainly unchanged ( < 10% variation) with use of pre- and postvaccination sera. In the CL assays PMNL alone as well as PMNL in the presence of unopsonized bacteria only gave background light emission.
4. Discussion The most relevant way of estimating protection against infection in a population following immunization with a bacterial vaccine is a clinical field trial. However, a field trial necessitates considerable amounts of time and resources. Reliable in vitro methods for estimating individual protection are therefore desirable although they cannot encounter the priming effect on the immune system induced by a vaccination. ELISA measures all antibodies directed to a particular antigen whereas their functional activity remains unknown. The concentration of serum antibodies that is enough to give protection against MC infection is not known exactly [16]. The protection level of anti-PS antibodies against Hib-infection has been suggested to lie between 0.1 and 1.O pg/ml [3 11. However, functional assays have shown a striking heterogenicity in functional activity of the Ig fraction following immunization against Hib-infection [ 14,32.33]. The variability has been related to differences in antibody specificity and avidity between and within Ig classes. For evaluation of the proportion of non-functional antibodies it is desirable to use functional assays such as complement-mediated bactericidal activity or opsonic activity in bacterial vaccine trials [14,34,34]. In these assays a human complement source is desirable since complement from other species is not as reliable [16]. The present investigation, of the opsonic and bactericidal activity induced by McA + C and Hib vaccination, in voluntary adults may be regarded as a pilot study. It was designed to compare and correlate the results of the functional assays with the concentration of serum antibodies measured by ELISA. Of special interest was to compare the results of serum opsonic Table 2 Coefficients
for
a linear
correlation
between
the
results
of the three
functional assays (CL-index, SBA and PK) and the total Ig fraction of anti-capsular serum antibodies against MC A C and Hlb measured with ELISA before vaccination (bold) and the coefficients for linear correlation tion sera
ELISA CL-index SBA
between (italics)
the fold
increase
of serum
CL-index
SBA
0.40
0.47 0.51
0.45
activity
in postvaccina-
PK 0.04 0.15
0.54 0.49
0.04 0.18
0.96 0.98
in Infrcrious
Disease
8 (1997)
213-220
activity measured by CL index and PK. The CL assay estimates the opsonizing activity of antibodies. The internal complement activity of the test sample is preserved in each experiment. The oxidative burst that is necessary to induce CL is dependent on the bacteriaPMNL interaction in the presence of opsonizing serum antibodies and complement. Thus. the bactericidal activity is not taken into account with the CL index technique. It is an indirect measurement of opsonophagocytosis without being in need of radioactive agents. The index calculation with the index- and control strains minimizes the risk of intra- and interassay variations though non-relevant PMNL-bacteria interaction may influence the results. The SBA and PK assays measure antibody-mediated bactericidal activity with preserved internal complement activity. By definition PK also takes the additional effect of opsonophagocytosis into consideration. They are robust and direct methods of bacterial killing but have the drawback of being laborious and time consuming. With the limitations of internal complement to dilute enough. the functional methods cannot discriminate bacterial killing between individuals with high antibody concentrations. This drawback reduces the validity of the tests, and this is more obvious with SBA and PK than with the CL-index technique. In the present study. the ELISA and the CL assay defined PRP-D as a better immunogen than PRPOMPc. The results presented in Fig. 2 also indicate that PRP-OMPc induces a less CL-index activity per pg anti-HibPS (ELISA) than PRP-D while the SBA and PK did not detect this qualitative difference. This may be due to a population of antibodies of lower avidity induced by PRP-OMPc, which is detected as reduced opsonic activity in the CL assay. The bactericidal activity of vaccine-induced antibodies, measured by SBA and PK. was the same for the two vaccines. This may suggest that there is no inter-vaccine difference of functional antibody properties such as avidity in conferring bactericidi, but it can also be a result of the inability of these assays to discriminate between the high serum activity that follows from immunization of adults by a conjugated Hib-vaccine. Furthermore, from the results of the PK assay there is no obvious way of estimating the proportion of opsonic activity because of the bactericidal interference. SBA is not as complex as PK and a bactericidal assay is most often adopted to detect functional activity of antibodies [6,14]. Qualitative differences in SBA, in contrast to the ELISA antibody results, were found in a recent trial of two MC A + C vaccines [36]. In McB vaccine trials there are reports of both poor [6] and good correlation [35] between serum concentrations of antibodies against various surface antigens of McB and serum bactericidal activity.
S. Hugosson
et al. : Serodtagnosis
and
Immunotl~erap~
In our study the mean response was good by all assays indicating that the generated antibodies acted functionally in most individuals. Interindividual differences in response characteristics existed but usually high antibody concentrations were associated with functional activity and vice versa. A possible explanation to poor functional activity despite high concentrations of antibodies is the prevalence of blocking antibodies, antibodies of low avidity or of non-functional Ig classes [14]. It has been suggested that IgA can inhibit the interaction between IgG and the bacteria which bind complement and mediate cell lysis or phagocytosis [32,37]. A proportionally high antibody response of IgA class to vaccination could be non-physiological in this respect. In our study one of the vaccines, who responded well in the ELISA but poor in the functional assays, had an exceptionally high IgA concentration in postvaccination serum (data not shown). The other non-responders had the equally high proportion of IgG/IgA ratio as the responders. The serum response measured with ELISA correlated better with CL than with the other two functional assays. This could imply that a main fraction of the antibodies generated by vaccination acted as opsonins. We found that the bacterial killing by PK in the presence of PMNL is somewhat but not significantly higher than in SBA suggesting that the activity measured with PK mainly is of bactericidal origin and the opsonizing properties induced by the vaccination probably are poorly considered. This suggestion is supported by the very strong correlation between the SBA and PK assays shown in Table 2. The advantage of Hib to Hint in resisting phagocytosis is shown in our CL experiment with prevaccination sera. In accordance with the specificity of antibodies induced by immunization, the CL activity against Hint and McB was unaffected in postvaccination sera following vaccination against Hib PS/McA + C, and these experiments can serve as negative controls. In future vaccine trials, an immunochemical calculation (ELISA or RIA) of serum concentrations of antibodies against relevant bacterial antigens is mandatory. With regard to the existence of antibodies that are reduced in specificity, of low avidity or of non-functional Ig class a functional assay can be complementary to make sure that an antibody response correlates with bactericidal or opsonic activity or passive protection in an animal model [23,34,36]. Functional assays are logical to use in trials with cellular vaccines such as McB outer membrane preparations, where the important antigens for protection are unknown. For measurements of serum opsonic activity the results of PK may be confusing because of the strong influence by the bactericidal activity. The CL index technique estimates this property in a more unaffected way. To evaluate the CL assay further it would be desirable to compare it
in lnjectious
Disease
8 (1997)
213-220
219
with other in vitro assays of opsonophagocytosis such as uptake of radioactive bacteria by PMNL and flow cytometry. It would also be of interest to correlate it with an animal model of passive protection.
Acknowledgements We wish to thank the Research Committee County Council for financial support.
of Grebro
References [II
Berg S, Trollfors B, Alestig K, Jodal U. Incidence, serogroups and case-fatality rate of invasive meningococcal infections in a Swedish region 197551989. Stand J Infect Dis 1992: 24: 333338. of PI Make12 PH, Takala AK. Peltola H, Eskola J. Epidemiology invasive Haemophilus injuenzae type b disease. J Infect Dis 1992; 165 (Suppl I): 2-6. S, Olcen P, Ekedahl C. Acute epiglottitis-etiology, [31 Hugosson epidemiology and outcome in a population before large-scale Haemophilus influenrae type b vaccination. Clin Otolaryngol 1994; 19: 44-5. MM, Christoffer NC. Griffiss JM, Baker CJ, Man141 Estabrook drel] RE. Sialylation and human neutrophil killing of group C Neissrria meningitidis. J Infect Dis 1992; 166: 1079988. basis of invasive H. irzjluenrae type b [51 Moxon ER. Molecular disease. J Infect Dis 1992; 165 (Suppl): 77781. [61 Milagres LG, Ramos RS, Sacchi CT. Melles CEA, Vieira VSD, Sato H. et al. Immune response of Brazilian children to Neisseria meningitidk serogroup B outer membrane protein vaccine: comparison of efficacy. J Infect Dis 1994; 62: 4419-24. A. Meningococcal disease and its control with meningo[71 Galazka coccal polysaccharide vaccines, Bull WHO 1982; 60: l-7. PI Murphy TV. White KE, Pastor P, Gabriel L, Medley F, Granoff DM. et al. Declining incidence of Haemophilus influenzar type b disease since introduction of vaccination. J Am Med Assoc 1993: 269: 24668. JK. Arnesen 0, Fredriksen JH, [91 Bjune G, Hoiby EA, Gronnesby Halstensen A. Effect of outer membrane vesicle vaccine against group B meningococcal disease in Norway). Lancet 1991; 338: 109336. DK, Baker CJ. 1101Griffiss J, Brandt BL, Broud DD, Gorofff Immune response of infants and children to disseminated infections with Neisseria meningitidis. J Infect Dis 1984; 150: 71-9. H, Hakulinen M. [‘II Eskola J. Peltola H. Takala AK, Kayhty Karanko V, et al. Efficacy of H. in@wtzar type b polysaccharide-diphtheria toxoid conjugate vaccine in infancy. N Engl J Med 1987; 317: 717-22. of complement source [I21 Zollinger WD. Mandrel] RE. Importance in bactericidal activity of human antibody and murine monoclonal antibody to meningococcal group B polysaccharide. Infect Immun 1983; 40: 257764. I. Gotschlich EC, Artenstein MS. Human immu1131Goldschneider nity to the meningococcus. I. The role of humoral antibodies. J Exp Med 1969: 129: 1307726. DM. Variability in the functional ['41 Amir J, Liang X, Granoff activity of vaccine-induced antibody to Harmophilus inJlurn:ae type b. Pediatr Res 1990; 27: 358-64. SL. Waldo B, Johnston RB. Separation of serum [I51 Newman bactericidal and opsonizing activities of Harmop/~ilus injuenzae type b. Infect lmmun 1973; 8: 164-6.
220 [I61
[17]
(IS]
[19]
[20]
[21]
[22]
[23]
[24]
[25] [26]
[27]
S. Hugosson
et a/.
Serodiagttosb
and Immurrofherap~
Fredlund H. Serum factors and polymorphonuclear leukocytes in human host defence against Neissrria mrningitidis. Studies of interactions with special reference to a chemiluminometric technique. Stand J Infect Dis 1993: SX7: l-72. Kaplan SL. Umstead CL. Mason EO. Anderson DC. Parke Jr JC, Feigin RD. Assessment of Huemophilus mfluenzar type b opsonins by neutrophil chemiluminescence. J Clin Microbial 1981: 13: 532-9. Fredlund H, Sellden H, Danielssom D, Olcen P. Early development in healthy children of serum opsonins against nonpathogenie Nrissrricr meningitidis. APM IS 1992; 100: 449- 54. Lehmann V, Solberg CO. Group B meningococci opsonins in serum measured by polymorphonuclear leukocyte chemiluminescence. APMIS 1980; X8: 227731. Guttormsen HK. Bjerknes R. Halstensen A. Naess A. H&by A. Solberg CO. Cross-reacting serum opsonins to meningococci after vaccination. J Infect Dis 1993; 167: 1314.-9. Halstensen A, Haneberg B. Froholm 0. Lehmann V, Frasch CE, Solberg CO. Human opsonins to meningococci after vaccination. Infect Immun 1984: 46: 67336. Crook G. Wilson R, Kroll S. Todd H. Garbett N. Moxon R. et al. Opsonic requirements and interaction of Harmophilus injluflltm:rre with human polymorphonuclear neutrophil leucocytes studied by luminol-enhanced chemiluminescence. Microb Pathog 19x9; 7: 101-10. Ojo-Amaize EA. Church JA. Barka NE, Agopian MS, Peter JB. A rapid and sensitive chemiluminescence assay for evaluation of functional opsonic activity of Htremophrlus influenxw type b specific antibodies. Clin Diagn Lab Immunol 1995; 2: 2X6-90. Fredlund H, Olcen P. Danielsson D. A reference procedure to study chemiluminescence induced in polymorphonuclear leukocytes by :Veisseria /rrrnirrgirit/i.c. APMIS 1988: 96: 941 -9. Boyum A. Separation of blood leukocytes, granulocytes and lymphocytes. Tissue Antigens 1974: 4: 269974. Arakere G. Frasch CE. Specificity of antibodies to O-acetyl-positive and 0-acetyl-negative group C meningococcal polysaccharides in sera from vaccinees and carriers. Infect Immun 1991; 59: 4349.. 56. Platonov AE, Beloborodov VB, Pavlota LI. Vershinina IV. Kayhty H. Vaccination of patients dehcient in a late complement
in Irtfectious
Disease
8 (1997)
213-220
component with tetravalent meningococcal capsular polysaccharide vaccine. Clin Exp Immunol 1995; 100: 32-9. S. Klyhty H. Saarinen L. Ronnberg P-R, Eskola J. [2X1Kurikka Immunity after one dose of Haemophilus in&n;ae type b conjugate vaccine. J Infect Dis 1995; 172: 1268-72. EC, Rey M. Triau R, Sparks KJ. Quantitative deter~291 Gotschlich minations of human immune response to immunization with meningococcal vaccines. J Clin Invest 1972; 51: 89996. JE. The effect of protein II and pili on the [301 Virji M, Heckels interaction of Nrisseria gonorrhoea with human polymorphonuclear leukocytes. J Gen Microbial 1986; 132: 503-12. DM, Holmes SJ. Comparative immunogenicity of [311 Granoff Haemophilu.c in/luen:ar type b polysaccharide-protein conjugate vaccines. Vaccine 1991; 9 (Suppl): 530.-4. JR, Barrus V, Cates KL, Siber CR. Functional charac[321 Schreiber terization of human IgG, IgM and IgA antibody directed to the capsule of Haemophilus iny%en;ae type b. J Infect Dis 1986; 153: 8-15. [33] Lucas AH, Granoff DM. Functional differences in idiotypically delined IgGl anti-polysaccharide antibodies elicited by vaccination with Haemopldus injurnzae type b polysaccharide protein conjugates. J Immunol 1995; 154: 41955202. DM. Lucas AH. Laboratory correlates of protection [341 Granoff against Haw~opldus in/?uen:ue type b disease. Importance of assessment of antibody avidity and immunologic memory. Ann NY Acad Sci 1995: 754: 278X88. A, Rosenqvist E, Pedersen AK, [351 Aase A, Bjune G. Hoiby Michaelsen T. Comparison among opsonic activity, antimeningococcal immunoglobulin G response and serum bactericidal activity against meningococci in sera from vaccinees after immunization with a serogroup B outer membrane vesicle vaccine. Infect Immun 1995: 63: 3531-6. JM, Chiu SS, Wong VK, Partridge S, Chang S, Chiu [361 Lieberman C et al. Safety and immunogenicity of a serogroups A/C Neisseriu mmingitidis oligosaccharide-protein conjugate vaccine in young children. J Am Med Assoc 1996; 275: 1499-503. J. Epidemic meningococcal disease: a synthesis of a [371 Grifliss hypothetical immunoepidemiologic model. Rev Infect Dis 1982; 4: 159 71.