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Immune response of mice to immunization with subunit influenza A vaccine in DTP vaccine
Vaccine, Vol. 13, No. 3, pp.2SS260, 1995 Copyright 0 1995 Elsevier Science Ltd Printed in Great Britain. All rights reserved 0264410x/95 $10.ao+0.cm
Immune response of mice to immunization with subunit influenza A vaccine in DTP vaccine Christopher
W. Potter
*$ ,
Hassan Tamizifar? and Roy Jennings?
Experiments were carried out to examine the initial feasibility of immunizing infants and children with inactivated influenza virus vaccine combined w.ithdiphtheria-tetanus-pertussis (DTP) vaccine. Groups of mice were immunized with saline vaccine or vaccine mixed with DTP: three doses of vaccine were given 3 weeks apart, and the antibody response and resistance to challenge infection were tested 3 weeks after the 1st and 3rd immunizations. The results showed that the antibody response and immunity to challenge virus infection were significantly greater for mice given vaccine in DTP than for mice given saline vaccine alone. Comparison of the response to graded doses of vaccine in saline or DTP indicated that vaccine in DTP was > 2.50-fold more eflective in inducing serum antibody and protection than saline vaccine alone. The enhancing activity of DTP was signljicant for the alum component alone; however, most of the adjuvant eflect was from the antigen components of the DTP vaccine. The results suggest that immunization against influenza in infants and young children could be achieved by combining small amounts of influenza antigen with DTP vaccines; however, the present results have been obtained in mice, and, since the responses to vaccines and adjuvants vary from species to species, the present results cannot be used to indicate similar @&Its in human volunteers. The results indicate the potential value of an immunization procedure which should be tested in volunteers and which could provide a simple strategy for the immunization of at-risk infants and children against influenza. Keywords
Influenza
A vaccine; diphtheria-tetanus-pertussis
vaccine;
immune
Numerous volunteer studies of inactivated influenza vaccines have shown iow levels of reactivity, induction of serum antibody and protection from challenge virus infection in 60-90% of vaccinees’. These vaccines are recommended for subjects for whom influenza infection is serious and life-threatening, including persons over 65 years of age, patients with chronic heart and chest disease, and patients with asthma, renal dysfunction and other metabolic disorders2: immunization of young children, particularly those with a history of recurrent chest infection or otitis media, is also recommended by some authorities3-‘. Arguments for a broader immunization policy for young children include the high incidence of infection and morbidity in this group at schools and crCches, and the replication of influenza virus to high titres has resulted in children being a focus of infection for others6q7. In contrast, arguments against a broader policy are that (i) the antibody responses of children are relatively poor due to a lack of earlier priming by live *Institute for Cancer Studies and ‘Department of Experimental and Clinical Microbiology, University of Sheffield Medical School, Beech Hill Road, Sheffield SlO 2RX, UK. ‘To whom correspondence should be addressed. (Received 27 January 1994; revised 20 May 1994; accepted 23 May 1994)
response
virus infection; (ii) higher reactivity rates indicate the use of split-virus vaccine, which is less immunogenic than whole-virus vaccines and thus requires two doses; (iii) the introduction of a policy of general immunization of children could interfere with other vaccine recommendations; and (iv) a low immunization rate has not been shown to be beneficial to the community’*‘. Many of these negative views could be countered if influenza immunization of children were combined with immunization against diphtheria, tetanus and whooping cough (DTP). Thus, separate immunization would not be required; DTP could enhance the immune response to influenza virus antigens, as shown in studies with influenza and other vaccines10-‘2; and an adjuvant effect of DTP may allow the concentration of virus proteins to be lowered to reduce reactions without impairing the immune response. Such a vaccine could provide protection for a vulnerable age group, and limit an important source of infection for other children and adults. To investigate the above proposal, an influenza A virus subunit vaccine was administered to mice in saline or combined with DTP vaccine, and the antibody responses and susceptibility to challenge virus infection were determined. In addition, subunit vaccine was given with
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Response to influenza
vaccine in DTP: C.W. Potter et al.
complete DTP, DTP antigens or alum separately to identify the adjuvant component of DTP. Finally, serial dilutions of subunit vaccine were inoculated into mice with and without DTP and the antibody response and susceptibility to challenge infection were measured to determine the extent of virus antigen sparing.
MATERIALS
dropwise with A/Beijing/89 virus at a concentration of lo2 MID,, in a 0.1 ml volume of PBS. Three days after challenge, mice were killed and nasal washings and lung specimens collected and tested for virus, as described above. Blood samples were taken and challenge studies were carried out at 21 days after one, two or three immunizations in the different experiments.
AND METHODS
Influenza virus and virus vaccine
Haemagglutination inhibition (HI) tests
Seed influenza virus A/Beijing/89 (H3N2) was kindly supplied by Dr T. Carstairs, Evans Medical, Speke, Liverpool, UK. Virus pools were prepared by inoculating 0.2 ml of a 10m3 dilution of seed virus into the allantoic cavity of lo-day-old embryonated eggs; after incubation at 37°C for 72 h, allantoic fluids were harvested, ampouled and stored at -70°C. Virus infectivity was determined by inoculating 0.1 ml of tenfold dilutions of virus in phosphate-buffered saline (PBS), pH 7.4, intranasally and dropwise into groups of Balb/c mice. After 72 h, nasal washings were collected and lungs were removed and processed to give a 40% (w/v) extract from each mouse: 0.2 ml of each undiluted sample was then inoculated allantoically into lo-day-old embryonated eggs, which were incubated for 72 h at 33°C before the allantoic fluids were removed and tested for virus by haemagglutination with fowl erythrocytes. From the results obtained for nasal washings, the 50% minimum infective dose (MID,,) was calculated. The influenza virus used for vaccine production was influenza virus A/Beijing X109, a reassortant of influenza viruses A/Beijing/353/89 (H3N2) and A/PR/8/34 (HlNl); the purified surface antigen vaccine (subunit vaccine) prepared from this virus was the influenza A (H3N2) component for incorporation into the commercial vaccine ‘Fluvirin’ for 1992/1993, and was kindly supplied by Evans Medical. The concentration of haemagglutinin (HA) in the subunit vaccine was calculated by single radial diffusion, as described previously13: standard reagents were kindly supplied by Dr J. Wood, National Institute of Biological Standards and Control, London, UK. Diphtheria, tetanus and whole-cell pertussis vaccine adsorbed to alum (DTP) was kindly supplied by Evans Medical: no attempt was made to standardize the DTP vaccine further. The complete DTP vaccine, alum and DTP antigens without alum were either complete vaccine or the vaccine components normally blended to give complete vaccine for human use, and were all used at a 1:4 dilution of the recommended human dose as this was the maximum dose tolerated by mice.
Prior to testing for HI antibody, sera were treated with cholera filtrate for 18 h at 37°C and subsequently heated at 56°C for 30 min to remove inhibitors. HI tests were carried out using a standard microtitre method with 4 HA units of virus14. Titres were expressed as the highest serum dilution which completely inhibited haemagglutination.
Animals Balb/c mice were obtained from a closed, randomly bred colony held at the University of Sheffield, UK. Mice were used at 8-9 weeks old when their weight was approximately 20-25 g.
Enzyme-linked immunosorbent assay (ELBA) The serum IgG and the nasal wash IgA antibody responses of mice to A/Beijing X109 subunit virus vaccine were measured using an ELISA procedure, as previously described”. Briefly, the wells of ELISA microplates were coated with 2OOpg of carbonated buffer, pH 9.6, containing 2.Opg HA of purified whole A/Beijing X109 virus, and left at 4°C for 24 h; the concentrated whole virus was kindly supplied by Evans Medical. After washing three times with 0.05% (v/v) Tween-20 in PBS, 200~1 samples of test or standard sera diluted 1:800 in PBS-Tween + 1% BSA were added in duplicate to appropriate wells. After 1 h incubation at 37°C the wells were again washed and 100~1 of goat anti-mouse IgG or anti-mouse IgA Fc-specific antibody, each conjugated to horseradish peroxidase, was added to the wells; these reagents were obtained from Nordic Immunology, Tilberg, The Netherlands, and the optimal concentrations were established by prior titration. After 1 h incubation at 37°C the plates were again washed and 1OOpg of O-phenol ethyldiamine substrate in citrate-phosphate buffer, pH 5.0, was added. After a further 30 min incubation in the dark at room temperature, the reaction was stopped by the addition of 2~ H,SO,, and the absorbance values were read at 490nm. For each set of tests, a positive control of known ELISA reading was included, and the results were normalized to this standard. To determine the concentration of IgG subclass antibody in mouse sera, similar tests were carried out using anti-mouse IgGl, IgG2a, IgG2b and IgG3 subclass antibodies; these were obtained from Sigma Chemicals. For each subclass, a subclass antibody preparation of known antibody concentration, obtained from Sigma Immunochemicals, was titrated in parallel, and the concentration of subclass antibody in mouse sera was calculated from absorbance values for control and test samples.
Experimental protocol Balb/c mice were test-bled and inoculated intramuscularly with one of the vaccines in a 0.225 ml volume: blood samples and nasal washings were collected at 21 days postimmunization. At this time, five or more mice from each group were inoculated intranasally and,
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Statistical analysis Comparisons between mouse groups with respect to the incidence and levels of serum antibody were made using the Mann-Whitney U test.
Response to influenza
RESULTS
To allow interpretation of the ELISA test values for antibody to influenza viral antigens, a panel of 48 mouse sera were titrated for antibody by both the HI and ELISA procedures; the latter tests were carried out at a single serum dilution of 1:BOO.The results are shown in Figure 1. Good correlation was observed for the two sets of data
Response of Balb/c mice to A/kijing X109 subunit vaccine Four groups of Balb/c mice were inoculated with 5.0 pug HA of A/Beijing X109 subunit vaccine mixed with PBS or DTP, or DTP or PBS alone; three immunizations were given at 3-week intervals, and the serum IgG antibody response at 3 weeks after each inoculation was measured by the ELISA technique. The results are given in Figure 2. For mice given saline vaccine alone, the ELISA absorbance value (mean + s.d.) after one inoculation was 0.268 fO.lO; this was equivalent to a mean HI titre of 1:40 (Figure I). The levels increased after the 2nd and 3rd inoculations to 0.940 + 0.295 and 2.322 + 0.019, respectively. In contrast, subunit vaccine in DTP induced significantly higher antibody responses: at 3 weeks following the 1st immunization, the ELISA reading was 1.417f0.081, and the reading was 2.834 (10.140) following the 3rd dose; these values were equivalent to HI titres > 1:3200. No detectable antibody was seen in the sera from animals given DTP or alum alone (Figure 2). The results indicate that DTP significantly enhanced the serum IgG antibody response to influenza subunit A/Beijing X109 vaccine following all three doses. From each group of Balb/c mice, five were inoculated intranasally with 100 MID,, of live A/Beijing/89 virus 3 weeks after the first vaccine inoculation. These results are shown in Table 1. Virus was recovered 3 days after inoculation from nasal washings from 4/5 and 3/5 mice immunized with alum or DTP alone; similar results were obtained for virus isolates from lung tissue from these animals. In contrast, some protection was seen for animals given a single dose of subunit vaccine; thus, virus was recovered from only two of five nasal washings and lungs from this group of animals. Virus was not
2. 0 ._
E
HI
mu
Correlation between HI titres and ELISA values
42
63
Time (days) Figure 2 Serum IgG antibody response at 21 days following immunization with DTP alone (+), PBS alone (m), subunit vaccine in DTP (0) or subunit vaccine in PBS (0). at days 0 (Vl), 21 (V2) and 42 (V3)
Table 1
al.
with an r value of 0.848. Previous studies have indicated that an HI antibody titre of approximately 40 can be equated with 50% immunity to challenge virus infection16, and this corresponded to an ELISA reading of 0.25; high HI titres 2 1:320 correlated with an ELISA reading > 0.75 (Figure I). In addition, antibody responses as measured by ELISA tests have been shown to correlate with protection against challenge virus infection’ 7.
Correlation of HI and ELISA tests for antibody to A/Beijing X109
Figure 1
vaccine in DTP: C.W. Potter et
immunity to challenge virus infection following immunization with influenza A/Beijing X109 subunit vaccine Response at 21 days (after one dose)
Response at 63 days (after three doses)*
Vaccine given (vehicle)a
No. of mice tested
Serum IgG antibody ELISA reading (mean+s.d.)
Subunit (PBS)
13
0.268+0.10
215
215
a
2.322*0.019
418
418
Subunit (DTP)
13
1.917~0.081
o/5
o/5
6
2.834kO.140
O/6
O/6
PBS (alum)
14
0.028kO.017
415
415
7
0.056kO.07
617
517
PBS (DTP)
14
0.062 i 0.031
315
415
5
0.106f0.011
515
415
Lung
No. of mice tested
Serum IgG antibody ELISA reading (mean&s.d.)
Nasal wash
Lung
No. of infections’ Nasal wash
No. of infections
‘Vaccine comprised 5~9 HA of A/Beijing X109 subunit vaccine in PBS or DTP, or PBS in alum or DTP ‘lmmunizations at days 0, 21 and 42 “Virus recovered from nasal wash or lung at 3 days postinfection with 100 MID, of A/Beijing189 virus
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1995 Volume
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Response to influenza
vaccine in DTP: C.W. Potter et al.
3.0
lgG3
2.0 1 .o
h
z
0
3
42
21
-
63
21
lgG2a
21
42
42
lgG2b
63
rime(Days)
21
42
Figure 3 Serum IgG subclass antibody responses at 21 days following the lst, 2nd and 3rd immunizations with 5pg HA of A/Beijing vaccine in saline (W) or in DTP (
recovered from either the lungs or the nasal washings of mice given a single inoculation of subunit vaccine in DTP (Table I).
Three weeks following the third vaccination, all remaining mice were inoculated intranasally with 100 MID5e of live virus. These results are shown in Table 1. Virus was recovered at 3 days postinoculation from nasal washings of 5/5 and 6/7 mice given three doses of DTP or alum alone, and from the lungs of 4/5 and 5/7 of the same mice, respectively. Virus was recovered from the nasal washings and lungs of 4/8 mice given three doses of subunit vaccine in saline. In contrast, virus was not recovered from the nasal washings or lungs of mice given three doses of subunit vaccine in DTP (Table 1). Thus, despite the relatively high level of serum IgG antibody in mice given three doses of subunit vaccine in saline (mean ELISA reading= 2.322, equivalent to an HI titre > 1:5000), half of these animals were susceptible to infection by challenge virus, whilst mice given subunit vaccine in DTP were completely refractile to challenge infection. The results suggest that the DTP subunit vaccine stimulated an immune response that was not seen in mice given subunit vaccine alone. IgG subclass antibody response of Balb/c mice to A/Beijing X109 subunit vaccine Serum samples from mice immunized with various antigen preparations, collected at 21 days following the lst, 2nd and 3rd immunizations, were tested for IgG subclass antibody to A/Beijing X109 virus using the ELISA test: all sera were tested at 1:800 dilution in
256
Vaccine 1995 Volume 13 Number 3
63
63 X109 subunit
parallel with standard antibody of known concentration, and the readings were converted to the concentration of subclass antibody. The results are shown in Figure 3. For mice given subunit vaccine in saline, the IgGl antibody concentration increased following each inoculation; however, only relatively small increments of subclass antibody were seen following each vaccine dose. Even smaller increments were seen for IgG2a, IgG2b and IgG3 subclass antibodies. In contrast, for mice given subunit vaccine in DTP, higher concentrations of IgGl were seen after the 1st vaccine dose, and larger increases in subclass antibody were detected after the 2nd and 3rd inoculations; relatively greater responses were seen for IgG2a and IgG2b, but the IgG3 response was small and comparable to that seen for mice given vaccine in saline (Figure 3). The results suggest a greater total IgG response and a broader subclass antibody response for mice given subunit vaccine in DTP compared with controls given vaccine in saline. Dose response to subunit vaccine To determine the dose response of Balb/c mice to influenza virus A/Beijing X109 subunit vaccine in saline or DTP, groups of mice were inoculated with fourfold dilutions of two vaccine formulations; the serum IgG antibody responses were measured 3 weeks after immunization at which time a 2nd dose was given, and again 3 weeks after the 2nd immunization. Susceptibility to challenge virus infection following two doses of vaccine was measured in all mice 3 weeks after the 2nd vaccine dose, as described above. The total IgG antibody
Response
in Saline
Subunil vaccine (@ml)
in DTP: C.W. Potter
et al.
of 5 pg HA in saline (Table 2). This result correlated with the serum IgG antibody responses, and indicated a 250-fold increase in protection against challenge virus infection for subunit vaccine in DTP.
responses to immunization are shown in Figure 4. Following the 1st dose of vaccine, the ELISA absorbance value (mean f s.d.) for mice given 5 pg of subunit vaccine in saline was 0.225+-0.019 which was equivalent to an HI titre of approximately 40, confirming the results shown in Figure 2: this result was boosted to 0.658 f 0.02 following the 2nd dose which was equivalent to an HI titre of approximately 240. A decreasing antibody response was seen for mice given a decreasing dose of subunit vaccine (Figure 4). In contrast, for mice given subunit vaccine in DTP, 0.02 pg of HA induced an ELISA antibody value of 0.844 ) 0.04 following the 1st dose and a value of 1.54 + 0.06 following reimmunization, equivalent to HI titres of 400 and > 5120, respectively. Thus, DTP amplified the antibody response to subunit vaccine > 250-fold. Following the two immunizations, all remaining animals were challenged intranasally with 100 MID,, of live influenza virus A/Beijing/89; the results are shown in Table 2. For mice given two doses of 5 pg HA in saline, significant protection was seen compared to control animals that received no vaccine; in contrast, no significant protection was seen in mice given two doses of 1.25 pg of HA or lower doses of saline vaccine. For mice given subunit vaccine in DTP, significant protection was seen following two doses of all vaccine dilutions, and the level of protection induced by two doses of 0.02pg HA in DTP was equivalent to that induced by two doses
Subunil vaccine (&ml)
to influenza vaccine
Adjuvant properties of DTP vaccine The adjuvant effect of DTP on the immune response to influenza A subunit vaccine could be due to the alum, the bacterial antigens or a combination of both. To determine the basis of this adjuvant effect, groups of Balb/c mice were inoculated with 5 pg of influenza A/Beijing X109 subunit vaccine in saline, with alum alone, with bacterial antigen components of DTP or with complete DTP vaccine: the concentration of alum, bacterial antigens or complete DTP was one-quarter of the recommended human dose in each case. Serum samples were collected 3 weeks after immunization, at which time a 2nd dose of vaccine was given: further blood samples were taken 3 weeks after the 2nd immunization and the animals were challenged with 100 MID,, of live influenza virus to determine the level of immunity. The results are shown in Table 3 and Figure 5. Following the 1st dose of vaccine, the serum IgG antibody response of mice given subunit vaccine in saline was 0.304rt 0.007 (Figure 5): this result was similar to that seen in earlier experiments (Figures 2 and 4). A significantly greater antibody response was seen for mice given subunit virus in alum, and a further significant increase was seen in mice given subunit vaccine in the bacterial antigen preparation: the highest value was seen for mice given subunit vaccine in complete DTP. Following the 2nd dose of vaccine, all antibody levels were boosted with the exception of mice given influenza vaccine in complete DTP; the level induced by two doses of vaccine in the bacterial antigen preparation exceeded that for mice given two doses of vaccine in complete DTP (Figure 5). Three weeks after the 2nd immunization, all mice were inoculated intranasally with 100 MID,, of live influenza A/Beijing/89 virus, and the results of virus isolation from lung and nasal washings 3 days after infection are shown in Table 3. Virus was recovered from nasal washes of all control mice given PBS alone and from some mice given subunit vaccine in sahne or in alum, but not from lungs or nasal washings of mice given subunit vaccine mixed with the bacterial antigen components of DTP or complete DTP. Thus, the enhanced antibody response and immunity seen for mice given subunit vaccine in
in DTP
Figure 4 Serum IgG antibody responses at 21 days following primary immunization (m) and reimmunization (m) of mice with graded doses of A/Beijing X109 subunit vaccine
Table 2 Serum IgG antibody response and protection against challenge virus infection following immunization with graded doses of A/Beijing X109 subunit vaccine Vaccine in DTP
Vaccine in saline Dose of A/Beijing x109 subunit vaccine
Serum IgG antibody ELISA reading (mean +s.d.)
No. of infections
(pg HA)
No. of mice tested
3 weeks after 1st immunization
3 weeks after 2nd immunization
Lung
5.0 1.25 0.30 0.08 0.02 Control
7 a 8 a 8 a
0.225f0.02 0.144kO.02 0.118iO.01 0.097f0.06 0.095 + 0.01 0.032 f 0.11
0.656 + 0.02 0.368+0.03 0.360 + 0.01 0.295 kO.02 0.240 kO.01 0.039+0.01
l/7 518 7/a 718 718 718
Serum IgG antibody ELISA reading (mean+s.d.)
No. of infections
Nasal wash
No. of mice tested
3 weeks after 1st immunization
3 weeks after 2nd immunization
Lung
Nasal wash
317
a
718
a
818 818 818 ala
a 8 a 7
1.556*0.08 1.54o_fo.o4 1.480+0.04 0.975 + 0.02 0.844+0.04 0.057f0.08
2.17a*o.ol 1.976t0.04 1.720 & 0.04 1.307f0.02 1.154rtO.06 0.057 + 0.01
018 018 l/8 l/8 118 617
018 tla 118 tla 418 617
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1995 Volume 13 Number 3
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Response to influenza vaccine in DTP:
C.W. Porter et al.
Table 3 Serum IgG antibody responses and protection against challenge virus infection following immunization with different formulations of A/Beijing X109 subunit vaccine After 1st immunization A/Beijing X109 subunit vaccine (SV) (5 pg HA)
sv + PBS SV + complete DTP SV+alum only SV+DTP less alum Control (PBS)
No. of infections Serum IgG antibody ELISA reading (mean+s.d.)
No. of mice tested
Serum IgG antibody ELISA reading (mean+s.d.)
Lung
Nasal wash
6 8 8 8 8
0.304f0.01 1.316+0.03 0.594*0.01 1.080 + 0.02 0.029 * 0.01
6 8 8 8 8
0.726 k 0.02 1.339 * 0.02 1.041 kO.02 1.498kO.01 0.025iO.01
116 O/8 l/8 O/8 718
216 018 418 O/8 818
1.2 1.0
2; 0.8
52 :Z WE $ J?.
0.6 0.4 0.2 0.0 PBS.
s.v+ PBS
s.v+ ALUM
S.V+DTP less alum
S.V+DTP complete
Figure 5 Serum IgG antibody responses at 21 days after primary immunization (m) and reimmunization (a) with 5pg HA subunit vaccine in alum, subunit vaccine with complete DTP, subunit vaccine with DTP less alum, subunit vaccine with PBS, or PBS alone
DTP was principally due to the presence of the bacterial antigens in the vaccine preparation.
DISCUSSION Although many authorities have indicated the value of immunization against influenza in infants and children with a history of recurrent respiratory tract infections or otitis mediajm5, others have not supported this suggestion for practical reasons ~3,’. Many of the objections could be overcome if immunization against influenza were incorporated into recommended vaccine policies, and the present study investigated one such strategy: (i) the simple mixture of influenza subunit vaccine with DTP vaccine combines two vaccines each shown to be safe for children18; (ii) the reported adjuvant effect of alum and Bordetella pertussis on the immune response could allow smaller quantities of influenza vaccine to be used, thus reducing further the reactogenicity of the vaccine”,‘g; (iii) adjuvants are reported to broaden the immune response which could enhance immunity to influenzazO; and (iv) it would not require a further inoculation and interference with recommended vaccine schedulesg. The present results show that incorporation of
258
Vaccine
(day 42)
No. of mice tested
1.61
g $
After 2nd immunization
(day 21)
1995 Volume
13 Number 3
influenza subunit vaccine into DTP enhanced the serum IgG response, broadened the IgG subclass antibody response, was antigen sparing, and increased the level of protection against challenge virus infection by a factor of 250 or more. Experiments were carried out with complete DTP combined with subunit vaccine dialysed to remove phosphates and allow adsorption of the viral antigens to the alum; however, the results observed (not shown) were similar to those seen for simple mixtures of virus vaccine and DTP, suggesting that the adjuvant effect of DTP is similar to that seen with muramyl dipeptides where physical association between antigen and adjuvant is not a requirement for effective adjuvant action’l. The adjuvant effects of DTP on the immune respol;ses to the influenza subunit vaccine were attributed mainly to the bacterial antigen components of DTP, particularly to the B. pertussis component as whole organisms of this species are known to have adjuvant activitylg. In addition, the stimulation of cell-mediated immune responses by DTP (see below) is probably due to this bacterial component, since alum is a poor or non-inducer of these responses ** . Because of possible reactivity in children to whole-cell B. pertussis, investigators are at present researching subcellular fractions of this organism for use in human vaccines: it is not known whether subcellular fractions would have the same adjuvant activity as whole organisms, and it is therefore suggested that some of the present experiments should be repeated using DTP containing subcellular fractions of B. pertussis. The present studies show that three doses of subunit vaccine in saline produced high levels of serum IgG antibody but failed to give complete protection against challenge virus infections: in contrast, no infections were seen following live virus challenge in mice given three doses of vaccine in DTP. The results suggest that the vaccine in DTP produced a more solid immunity, probably by broadening the immune responses and by induction of immune parameters not induced by saline vaccine. This effect cannot be attributed to production of local antibody, which as been shown to be important in the induction of immunity23324: the present studies indicate that both saline- and DTP-formulated vaccines induced relatively low and similar levels of local IgA antibody (data not shown). Possibly more important is the induction of IgG subclass antibodies: saline vaccine principally induced an IgGl antibody response, whilst vaccine in DTP induced a marked increase in IgG2a and IgG2b antibody. Previous studies have shown that the
Response to influenza
production of TgG2a antibodies is associated with the production of interleukin-2 and interferon-y*‘; these cytokines are secreted by Thi-I subset T lymphocytes26,27 which mediate a range of immune responses, including neutralizing activity**, complement activation29, phagocytosis3’, and cell-mediated immune responses31. Thus, it is possible that the enhanced immunity induced by the subunit vaccine in DTP is due to broader immune responses. The present results have been obtained in experiments in mice and cannot be extrapolated to man: immune reactions to vaccines and adjuvant effects are recognized to differ from species to species3*. The present results were obtained with a different adjuvant system to those used previously which further limits extrapolation. However, the results suggest that incorporation of subunit influenza A vaccine into DTP may have many advantages as listed above, and indicate that studies should now be carried out in human subjects. Apart from the enhanced immunity observed in mice given subunit influenza vaccine in DTP, the formulation was antigen sparing: this offers a potential advantage in time of vaccine shortage and a further reduction in the influenza vaccine reactivity that occurs due to the innate toxicity of the virus haemagglutinin33. Should DTP be shown to enhance the immune response and protection against influenza in humans, the results offer a simple strategy for the immunization of at-risk infants and children to control infection: a wider strategy could limit infection in this age group which is suggested to be a major source of infection for other children and for adults. There is also potential for production of a less reactive influenza vaccine preparation for adults.
7
a
9
10
11
12
13
14
15
16 17
18
ACKNOWLEDGEMENTS C.W.P. is supported by the Yorkshire Cancer Research Campaign. The authors are grateful for the generous gift of influenza A/Beijing X109 virus, subunit vaccine and DTP vaccine from Dr J. Furminger and Dr T. Colgate, Evans Medical, Speke, Liverpool, UK, and the expert technical assistance of Mrs T. Smith.
19
20
21 22 23
REFERENCES Potter, C.W. Inactivated influenza virus vaccine. In: Basic and Applied Influenza Research (Ed. Beare, AS.), CRC Press, Boca Raton, Florida, 1982, pp. 119-158 Prevention and Control of Influenza. Recommendations for the lmmunization Practices Advisory Committee (ACIP). MMWR 1992, 41, l-17 Glezen. W.P., Paredes, A. and Taber, L.H. Influenza in children. Relationship to other respiratory agents. JAMA 1986,243,1345-1349 Heikkinen, T., Rouskanen, O., Waris, M., Ziegler, T., Arola, M. and Halonen, P. Influenza vaccination in the prevention of acute otitis media in children. Am. J. Dis. Child. 1991, 145, 445-448 Glezen,W.P., Taber, L.H., Gruber, W.C., Piedra, P.A., Clover, R.D., Demmler, R.W. et al. Family studies of vaccine efficacy in children: comparison of protection provided by inactivated and attenuated influenza vaccines. In: Oprions for the Control of Influenza, volume II (Eds Hannoun, C., Kendal, A.P., Klenk, H.D. and Ruben, F.L.) Excerpta Medica, Amsterdam, 1993, pp. 435-437 Glezen, W.P. Considerations of the risk of influenza in children and indications for prophylaxis. Rev. Infect. Dis. 1980, 2, 40
24
25
2%
27
28
vaccine in DTP: C. W. Potter et al.
Hall, C.E., Cooney. M.K. and Fox, J.P. The Seattle virus watch. IV. Comparative epidemiological observations of infections with influenza A and B viruses, 1965-1969, in families with young children. Am. J. fpidemiol. 1973, 98, 366-386 Nicholson, K.G., Tyrrell, D.A.J., Harrison, P., Potter, C.W., Jennings, R., Clark, A. et a/. Clinical studies of monovalent inactivated whole virus and subunit A/USSR/77 (HlNl) vaccine: serological responses and clinical reactions. J. Biol. Stand. 1979, 7, 123-136 Potter, C.W. Vaccinations of infants and children: conclusions and recommendations. In: Options for the Control of Influenza, volume II (Eds Hannoun, C., Kendal, A.P., Klenk, H.D. and Ruben, F.L.) Excerpta Medica, Amsterdam, 1993, pp. 449-456 Hennessy, A.V. and Davenport, F.M. Studies on vaccination of infants against influenza with haemagglutinin. Proc. Sot. Exp. Biol. Med. 1974,146, 206-204 Watemberg, N., Dagan, R., Arbelli, I., Belmaker, I., Morag, A. and Hessel, L. Safety and immunogenicity of Haemophilus type b tetanus protein conjugate vaccine, mixed in the same syringe with diphtheria-tetanus-pertussis vaccine in young infants. Pediatr. Infect. Dis. J. 1991, 10, 758-763 Barra, A., Dagan, R., Preud’homme, J.L., Bajart, A., Danve, B. and Fritzel, B. Characterization of the serum antibody response by Haemophilus influenza type b tetanus protein conjugate vaccine in infants receiving a DTP-combined vaccine from 2 months of age. Vaccine 1993,ll. 1003-1006 Al-Khayatt. R., Jennings, R. and Potter, C.W. Interpretation of responses and protective levels of antibody against attenuated influenza A viruses using single radial haemolysis. J. Hyg. (Camb.) 1984, 92, 301-312 Jennings, R., Smith, T.L., Spencer, R.C., Mellersh, A.M., Edey, D., Fenton, P. et al. Inactivated influenza vaccines in man: a comparative study of subunit and split vaccines using two methods of assessment of antibody responses. Vaccine 1984, 2, 75-86 Ben Ahmeida, E.T., Jennings, R., Erturk, M. and Potter, C.W. The IgA and subclass IgG response and protection in mice immunised with influenza antigens administered as ISCOMs, with FCA. ALH or as infectious virus. Arch. Viral. 1992, 125. 71-86 Potter, C.W. and Oxford, J.S. Determinants of immunity to influenza infections in man. Br. Med. Bull. 1979, 3.5, 69-75 Webster, R.G. and Thomas, T.L. Efficacy of equine influenza vaccines for protection against AIEquinelJilinl89 (H3N8) - a new equine influenza virus. Vaccine 1993, 11, 987-991 Miles, R., Potter, C.W., Clark, A. and Jennings, R. A comparative study of the reactogenicity and immunogenicity of two inactivated influenza vaccines in children. J. Biol. Stand. 1982, 10, 59-68 Dresser, D.W., Works, H.H. and Anderson, H.R. The effect of pertussis vaccine on the immune response of mice to sheep red blood cells. C/in. fxp. Immunol. 1971, 7, 817-831 Halman, C. New approaches to vaccine delivery. In: Opfions for the Control of Influenza, volume II (Eds Hannoun, C., Kendal, A.P., Klenk, H.D. and Ruben, F.L.) Excerpta Medica, Amsterdam, 1993, pp. 389-391 Lecerc, C. and Vogel, F. Synthetic immunomodulators and synthetic vaccines. Crit. Rev. Thera. Drug Carrier Syst. 1986, 2, 353-369 Bomford, R. The comparative selectivity of adjuvants for humoral and cell-mediated immunity. C/in. Exp. Immunol. 1980,39,435-448 Freestone, D.S., Hamilton-Smith, S., Schild, G.C., Buckman, R., Chinn, S. and Tyrrell, D.A.J. Antibody responses and resistance to challenge in volunteers vaccinated with live attenuated, detergent split and oil adjuvant AZ/Hong Kong/68 (H3N2) influenza vaccines. J. Hyg. (Camb.) 1972, 70, 531-543 Clements, M.L., Betts, R.F., Tierney, E.L. and Murphy, B.R. Serum and nasal wash antibodies associated with resistance to experimental challenge with influenza A wild-type virus. J. C/in. Microbial. 1986, 24, 157-160 Snapper, CM. and Paul, W.E. Interferon 7 and B cell stimulatory factor-l reciprocally regulate immunoglobulin isotype control. Science 1987, 2.36, 944-947 Mosmann, T.R., Cherwinksi, H.M., Bond, M.W.. Geidlin, M.A. and Coffman. R.L. Two types of murine helper T cell clone. 1. Definition according to profiles of lymphokine activities and secreted proteins. J. Immunol. 1986,136, 2348-2357 KurtJones, E.A., Hambert, S., O’Hara, J.. Paul, WE. and Abbas, A.K. Heterogeneity of helper/inducer T lymphoctes. 1. Lymphokine production and lymphokine responsiveness. J. Exp. Med. 1987,166, 1774-1787 Hocart. M.J., Mackenzie, J.S. and Stewart, G.A. The immunoglobulin G subclass responses of mice to influenza A virus: the effect of mouse strain, and the neutralising activities of individual protein-
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A-purified subclass antibodies. J. Gen. Viral. 1989, 70, 2434-2448 Spiegelberg. H.L. Biological activities of immunoglobulin of different classes and subclasses. Rev. Immunol. 1974.19, 25%B4 Weiner, E. The ability of IgG subclasses to cause the elimination of targets in vivo and to mediate their destruction by phagocytosis in vitro. In: The human IgG subclass: molecule analysis of structure, function and regulation (Ed. Shakib, F.) Pergamon Press, Oxford, 1990, pp. 135-160 Cher, D.J. and Mosmann, T.R. Two types of helper T cell clone.
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2. Delayed type hypersensitivity is mediated by THl clones. J. Immunol. 1997,138,3@84694 Davenport, F.M. Antigenic enhancement of ether-extracted influenza virus vaccines by AIPO.,. Proc. Sot. Exp. Biol. Med. 1968, 127, 557-590 Pickering, J.M.. Smith, H. and Sweet, C. Influenza virus pyrogenicity: central role of structural orientation of virion components and involvement of viral lipid and glycoproteins. J. Gen. Viral. 1992, 73,1345-1354
Immune response of mice to immunization with subunit influenza A vaccine in DTP vaccine Christopher
W. Potter
*$ ,
Hassan Tamizifar? and Roy Jennings?
Experiments were carried out to examine the initial feasibility of immunizing infants and children with inactivated influenza virus vaccine combined w.ithdiphtheria-tetanus-pertussis (DTP) vaccine. Groups of mice were immunized with saline vaccine or vaccine mixed with DTP: three doses of vaccine were given 3 weeks apart, and the antibody response and resistance to challenge infection were tested 3 weeks after the 1st and 3rd immunizations. The results showed that the antibody response and immunity to challenge virus infection were significantly greater for mice given vaccine in DTP than for mice given saline vaccine alone. Comparison of the response to graded doses of vaccine in saline or DTP indicated that vaccine in DTP was > 2.50-fold more eflective in inducing serum antibody and protection than saline vaccine alone. The enhancing activity of DTP was signljicant for the alum component alone; however, most of the adjuvant eflect was from the antigen components of the DTP vaccine. The results suggest that immunization against influenza in infants and young children could be achieved by combining small amounts of influenza antigen with DTP vaccines; however, the present results have been obtained in mice, and, since the responses to vaccines and adjuvants vary from species to species, the present results cannot be used to indicate similar @&Its in human volunteers. The results indicate the potential value of an immunization procedure which should be tested in volunteers and which could provide a simple strategy for the immunization of at-risk infants and children against influenza. Keywords
Influenza
A vaccine; diphtheria-tetanus-pertussis
vaccine;
immune
Numerous volunteer studies of inactivated influenza vaccines have shown iow levels of reactivity, induction of serum antibody and protection from challenge virus infection in 60-90% of vaccinees’. These vaccines are recommended for subjects for whom influenza infection is serious and life-threatening, including persons over 65 years of age, patients with chronic heart and chest disease, and patients with asthma, renal dysfunction and other metabolic disorders2: immunization of young children, particularly those with a history of recurrent chest infection or otitis media, is also recommended by some authorities3-‘. Arguments for a broader immunization policy for young children include the high incidence of infection and morbidity in this group at schools and crCches, and the replication of influenza virus to high titres has resulted in children being a focus of infection for others6q7. In contrast, arguments against a broader policy are that (i) the antibody responses of children are relatively poor due to a lack of earlier priming by live *Institute for Cancer Studies and ‘Department of Experimental and Clinical Microbiology, University of Sheffield Medical School, Beech Hill Road, Sheffield SlO 2RX, UK. ‘To whom correspondence should be addressed. (Received 27 January 1994; revised 20 May 1994; accepted 23 May 1994)
response
virus infection; (ii) higher reactivity rates indicate the use of split-virus vaccine, which is less immunogenic than whole-virus vaccines and thus requires two doses; (iii) the introduction of a policy of general immunization of children could interfere with other vaccine recommendations; and (iv) a low immunization rate has not been shown to be beneficial to the community’*‘. Many of these negative views could be countered if influenza immunization of children were combined with immunization against diphtheria, tetanus and whooping cough (DTP). Thus, separate immunization would not be required; DTP could enhance the immune response to influenza virus antigens, as shown in studies with influenza and other vaccines10-‘2; and an adjuvant effect of DTP may allow the concentration of virus proteins to be lowered to reduce reactions without impairing the immune response. Such a vaccine could provide protection for a vulnerable age group, and limit an important source of infection for other children and adults. To investigate the above proposal, an influenza A virus subunit vaccine was administered to mice in saline or combined with DTP vaccine, and the antibody responses and susceptibility to challenge virus infection were determined. In addition, subunit vaccine was given with
Vaccine 19% Volume 13 Number 3
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Response to influenza
vaccine in DTP: C.W. Potter et al.
complete DTP, DTP antigens or alum separately to identify the adjuvant component of DTP. Finally, serial dilutions of subunit vaccine were inoculated into mice with and without DTP and the antibody response and susceptibility to challenge infection were measured to determine the extent of virus antigen sparing.
MATERIALS
dropwise with A/Beijing/89 virus at a concentration of lo2 MID,, in a 0.1 ml volume of PBS. Three days after challenge, mice were killed and nasal washings and lung specimens collected and tested for virus, as described above. Blood samples were taken and challenge studies were carried out at 21 days after one, two or three immunizations in the different experiments.
AND METHODS
Influenza virus and virus vaccine
Haemagglutination inhibition (HI) tests
Seed influenza virus A/Beijing/89 (H3N2) was kindly supplied by Dr T. Carstairs, Evans Medical, Speke, Liverpool, UK. Virus pools were prepared by inoculating 0.2 ml of a 10m3 dilution of seed virus into the allantoic cavity of lo-day-old embryonated eggs; after incubation at 37°C for 72 h, allantoic fluids were harvested, ampouled and stored at -70°C. Virus infectivity was determined by inoculating 0.1 ml of tenfold dilutions of virus in phosphate-buffered saline (PBS), pH 7.4, intranasally and dropwise into groups of Balb/c mice. After 72 h, nasal washings were collected and lungs were removed and processed to give a 40% (w/v) extract from each mouse: 0.2 ml of each undiluted sample was then inoculated allantoically into lo-day-old embryonated eggs, which were incubated for 72 h at 33°C before the allantoic fluids were removed and tested for virus by haemagglutination with fowl erythrocytes. From the results obtained for nasal washings, the 50% minimum infective dose (MID,,) was calculated. The influenza virus used for vaccine production was influenza virus A/Beijing X109, a reassortant of influenza viruses A/Beijing/353/89 (H3N2) and A/PR/8/34 (HlNl); the purified surface antigen vaccine (subunit vaccine) prepared from this virus was the influenza A (H3N2) component for incorporation into the commercial vaccine ‘Fluvirin’ for 1992/1993, and was kindly supplied by Evans Medical. The concentration of haemagglutinin (HA) in the subunit vaccine was calculated by single radial diffusion, as described previously13: standard reagents were kindly supplied by Dr J. Wood, National Institute of Biological Standards and Control, London, UK. Diphtheria, tetanus and whole-cell pertussis vaccine adsorbed to alum (DTP) was kindly supplied by Evans Medical: no attempt was made to standardize the DTP vaccine further. The complete DTP vaccine, alum and DTP antigens without alum were either complete vaccine or the vaccine components normally blended to give complete vaccine for human use, and were all used at a 1:4 dilution of the recommended human dose as this was the maximum dose tolerated by mice.
Prior to testing for HI antibody, sera were treated with cholera filtrate for 18 h at 37°C and subsequently heated at 56°C for 30 min to remove inhibitors. HI tests were carried out using a standard microtitre method with 4 HA units of virus14. Titres were expressed as the highest serum dilution which completely inhibited haemagglutination.
Animals Balb/c mice were obtained from a closed, randomly bred colony held at the University of Sheffield, UK. Mice were used at 8-9 weeks old when their weight was approximately 20-25 g.
Enzyme-linked immunosorbent assay (ELBA) The serum IgG and the nasal wash IgA antibody responses of mice to A/Beijing X109 subunit virus vaccine were measured using an ELISA procedure, as previously described”. Briefly, the wells of ELISA microplates were coated with 2OOpg of carbonated buffer, pH 9.6, containing 2.Opg HA of purified whole A/Beijing X109 virus, and left at 4°C for 24 h; the concentrated whole virus was kindly supplied by Evans Medical. After washing three times with 0.05% (v/v) Tween-20 in PBS, 200~1 samples of test or standard sera diluted 1:800 in PBS-Tween + 1% BSA were added in duplicate to appropriate wells. After 1 h incubation at 37°C the wells were again washed and 100~1 of goat anti-mouse IgG or anti-mouse IgA Fc-specific antibody, each conjugated to horseradish peroxidase, was added to the wells; these reagents were obtained from Nordic Immunology, Tilberg, The Netherlands, and the optimal concentrations were established by prior titration. After 1 h incubation at 37°C the plates were again washed and 1OOpg of O-phenol ethyldiamine substrate in citrate-phosphate buffer, pH 5.0, was added. After a further 30 min incubation in the dark at room temperature, the reaction was stopped by the addition of 2~ H,SO,, and the absorbance values were read at 490nm. For each set of tests, a positive control of known ELISA reading was included, and the results were normalized to this standard. To determine the concentration of IgG subclass antibody in mouse sera, similar tests were carried out using anti-mouse IgGl, IgG2a, IgG2b and IgG3 subclass antibodies; these were obtained from Sigma Chemicals. For each subclass, a subclass antibody preparation of known antibody concentration, obtained from Sigma Immunochemicals, was titrated in parallel, and the concentration of subclass antibody in mouse sera was calculated from absorbance values for control and test samples.
Experimental protocol Balb/c mice were test-bled and inoculated intramuscularly with one of the vaccines in a 0.225 ml volume: blood samples and nasal washings were collected at 21 days postimmunization. At this time, five or more mice from each group were inoculated intranasally and,
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Vaccine 1995 Volume 13 Number 3
Statistical analysis Comparisons between mouse groups with respect to the incidence and levels of serum antibody were made using the Mann-Whitney U test.
Response to influenza
RESULTS
To allow interpretation of the ELISA test values for antibody to influenza viral antigens, a panel of 48 mouse sera were titrated for antibody by both the HI and ELISA procedures; the latter tests were carried out at a single serum dilution of 1:BOO.The results are shown in Figure 1. Good correlation was observed for the two sets of data
Response of Balb/c mice to A/kijing X109 subunit vaccine Four groups of Balb/c mice were inoculated with 5.0 pug HA of A/Beijing X109 subunit vaccine mixed with PBS or DTP, or DTP or PBS alone; three immunizations were given at 3-week intervals, and the serum IgG antibody response at 3 weeks after each inoculation was measured by the ELISA technique. The results are given in Figure 2. For mice given saline vaccine alone, the ELISA absorbance value (mean + s.d.) after one inoculation was 0.268 fO.lO; this was equivalent to a mean HI titre of 1:40 (Figure I). The levels increased after the 2nd and 3rd inoculations to 0.940 + 0.295 and 2.322 + 0.019, respectively. In contrast, subunit vaccine in DTP induced significantly higher antibody responses: at 3 weeks following the 1st immunization, the ELISA reading was 1.417f0.081, and the reading was 2.834 (10.140) following the 3rd dose; these values were equivalent to HI titres > 1:3200. No detectable antibody was seen in the sera from animals given DTP or alum alone (Figure 2). The results indicate that DTP significantly enhanced the serum IgG antibody response to influenza subunit A/Beijing X109 vaccine following all three doses. From each group of Balb/c mice, five were inoculated intranasally with 100 MID,, of live A/Beijing/89 virus 3 weeks after the first vaccine inoculation. These results are shown in Table 1. Virus was recovered 3 days after inoculation from nasal washings from 4/5 and 3/5 mice immunized with alum or DTP alone; similar results were obtained for virus isolates from lung tissue from these animals. In contrast, some protection was seen for animals given a single dose of subunit vaccine; thus, virus was recovered from only two of five nasal washings and lungs from this group of animals. Virus was not
2. 0 ._
E
HI
mu
Correlation between HI titres and ELISA values
42
63
Time (days) Figure 2 Serum IgG antibody response at 21 days following immunization with DTP alone (+), PBS alone (m), subunit vaccine in DTP (0) or subunit vaccine in PBS (0). at days 0 (Vl), 21 (V2) and 42 (V3)
Table 1
al.
with an r value of 0.848. Previous studies have indicated that an HI antibody titre of approximately 40 can be equated with 50% immunity to challenge virus infection16, and this corresponded to an ELISA reading of 0.25; high HI titres 2 1:320 correlated with an ELISA reading > 0.75 (Figure I). In addition, antibody responses as measured by ELISA tests have been shown to correlate with protection against challenge virus infection’ 7.
Correlation of HI and ELISA tests for antibody to A/Beijing X109
Figure 1
vaccine in DTP: C.W. Potter et
immunity to challenge virus infection following immunization with influenza A/Beijing X109 subunit vaccine Response at 21 days (after one dose)
Response at 63 days (after three doses)*
Vaccine given (vehicle)a
No. of mice tested
Serum IgG antibody ELISA reading (mean+s.d.)
Subunit (PBS)
13
0.268+0.10
215
215
a
2.322*0.019
418
418
Subunit (DTP)
13
1.917~0.081
o/5
o/5
6
2.834kO.140
O/6
O/6
PBS (alum)
14
0.028kO.017
415
415
7
0.056kO.07
617
517
PBS (DTP)
14
0.062 i 0.031
315
415
5
0.106f0.011
515
415
Lung
No. of mice tested
Serum IgG antibody ELISA reading (mean&s.d.)
Nasal wash
Lung
No. of infections’ Nasal wash
No. of infections
‘Vaccine comprised 5~9 HA of A/Beijing X109 subunit vaccine in PBS or DTP, or PBS in alum or DTP ‘lmmunizations at days 0, 21 and 42 “Virus recovered from nasal wash or lung at 3 days postinfection with 100 MID, of A/Beijing189 virus
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1995 Volume
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255
Response to influenza
vaccine in DTP: C.W. Potter et al.
3.0
lgG3
2.0 1 .o
h
z
0
3
42
21
-
63
21
lgG2a
21
42
42
lgG2b
63
rime(Days)
21
42
Figure 3 Serum IgG subclass antibody responses at 21 days following the lst, 2nd and 3rd immunizations with 5pg HA of A/Beijing vaccine in saline (W) or in DTP (
recovered from either the lungs or the nasal washings of mice given a single inoculation of subunit vaccine in DTP (Table I).
Three weeks following the third vaccination, all remaining mice were inoculated intranasally with 100 MID5e of live virus. These results are shown in Table 1. Virus was recovered at 3 days postinoculation from nasal washings of 5/5 and 6/7 mice given three doses of DTP or alum alone, and from the lungs of 4/5 and 5/7 of the same mice, respectively. Virus was recovered from the nasal washings and lungs of 4/8 mice given three doses of subunit vaccine in saline. In contrast, virus was not recovered from the nasal washings or lungs of mice given three doses of subunit vaccine in DTP (Table 1). Thus, despite the relatively high level of serum IgG antibody in mice given three doses of subunit vaccine in saline (mean ELISA reading= 2.322, equivalent to an HI titre > 1:5000), half of these animals were susceptible to infection by challenge virus, whilst mice given subunit vaccine in DTP were completely refractile to challenge infection. The results suggest that the DTP subunit vaccine stimulated an immune response that was not seen in mice given subunit vaccine alone. IgG subclass antibody response of Balb/c mice to A/Beijing X109 subunit vaccine Serum samples from mice immunized with various antigen preparations, collected at 21 days following the lst, 2nd and 3rd immunizations, were tested for IgG subclass antibody to A/Beijing X109 virus using the ELISA test: all sera were tested at 1:800 dilution in
256
Vaccine 1995 Volume 13 Number 3
63
63 X109 subunit
parallel with standard antibody of known concentration, and the readings were converted to the concentration of subclass antibody. The results are shown in Figure 3. For mice given subunit vaccine in saline, the IgGl antibody concentration increased following each inoculation; however, only relatively small increments of subclass antibody were seen following each vaccine dose. Even smaller increments were seen for IgG2a, IgG2b and IgG3 subclass antibodies. In contrast, for mice given subunit vaccine in DTP, higher concentrations of IgGl were seen after the 1st vaccine dose, and larger increases in subclass antibody were detected after the 2nd and 3rd inoculations; relatively greater responses were seen for IgG2a and IgG2b, but the IgG3 response was small and comparable to that seen for mice given vaccine in saline (Figure 3). The results suggest a greater total IgG response and a broader subclass antibody response for mice given subunit vaccine in DTP compared with controls given vaccine in saline. Dose response to subunit vaccine To determine the dose response of Balb/c mice to influenza virus A/Beijing X109 subunit vaccine in saline or DTP, groups of mice were inoculated with fourfold dilutions of two vaccine formulations; the serum IgG antibody responses were measured 3 weeks after immunization at which time a 2nd dose was given, and again 3 weeks after the 2nd immunization. Susceptibility to challenge virus infection following two doses of vaccine was measured in all mice 3 weeks after the 2nd vaccine dose, as described above. The total IgG antibody
Response
in Saline
Subunil vaccine (@ml)
in DTP: C.W. Potter
et al.
of 5 pg HA in saline (Table 2). This result correlated with the serum IgG antibody responses, and indicated a 250-fold increase in protection against challenge virus infection for subunit vaccine in DTP.
responses to immunization are shown in Figure 4. Following the 1st dose of vaccine, the ELISA absorbance value (mean f s.d.) for mice given 5 pg of subunit vaccine in saline was 0.225+-0.019 which was equivalent to an HI titre of approximately 40, confirming the results shown in Figure 2: this result was boosted to 0.658 f 0.02 following the 2nd dose which was equivalent to an HI titre of approximately 240. A decreasing antibody response was seen for mice given a decreasing dose of subunit vaccine (Figure 4). In contrast, for mice given subunit vaccine in DTP, 0.02 pg of HA induced an ELISA antibody value of 0.844 ) 0.04 following the 1st dose and a value of 1.54 + 0.06 following reimmunization, equivalent to HI titres of 400 and > 5120, respectively. Thus, DTP amplified the antibody response to subunit vaccine > 250-fold. Following the two immunizations, all remaining animals were challenged intranasally with 100 MID,, of live influenza virus A/Beijing/89; the results are shown in Table 2. For mice given two doses of 5 pg HA in saline, significant protection was seen compared to control animals that received no vaccine; in contrast, no significant protection was seen in mice given two doses of 1.25 pg of HA or lower doses of saline vaccine. For mice given subunit vaccine in DTP, significant protection was seen following two doses of all vaccine dilutions, and the level of protection induced by two doses of 0.02pg HA in DTP was equivalent to that induced by two doses
Subunil vaccine (&ml)
to influenza vaccine
Adjuvant properties of DTP vaccine The adjuvant effect of DTP on the immune response to influenza A subunit vaccine could be due to the alum, the bacterial antigens or a combination of both. To determine the basis of this adjuvant effect, groups of Balb/c mice were inoculated with 5 pg of influenza A/Beijing X109 subunit vaccine in saline, with alum alone, with bacterial antigen components of DTP or with complete DTP vaccine: the concentration of alum, bacterial antigens or complete DTP was one-quarter of the recommended human dose in each case. Serum samples were collected 3 weeks after immunization, at which time a 2nd dose of vaccine was given: further blood samples were taken 3 weeks after the 2nd immunization and the animals were challenged with 100 MID,, of live influenza virus to determine the level of immunity. The results are shown in Table 3 and Figure 5. Following the 1st dose of vaccine, the serum IgG antibody response of mice given subunit vaccine in saline was 0.304rt 0.007 (Figure 5): this result was similar to that seen in earlier experiments (Figures 2 and 4). A significantly greater antibody response was seen for mice given subunit virus in alum, and a further significant increase was seen in mice given subunit vaccine in the bacterial antigen preparation: the highest value was seen for mice given subunit vaccine in complete DTP. Following the 2nd dose of vaccine, all antibody levels were boosted with the exception of mice given influenza vaccine in complete DTP; the level induced by two doses of vaccine in the bacterial antigen preparation exceeded that for mice given two doses of vaccine in complete DTP (Figure 5). Three weeks after the 2nd immunization, all mice were inoculated intranasally with 100 MID,, of live influenza A/Beijing/89 virus, and the results of virus isolation from lung and nasal washings 3 days after infection are shown in Table 3. Virus was recovered from nasal washes of all control mice given PBS alone and from some mice given subunit vaccine in sahne or in alum, but not from lungs or nasal washings of mice given subunit vaccine mixed with the bacterial antigen components of DTP or complete DTP. Thus, the enhanced antibody response and immunity seen for mice given subunit vaccine in
in DTP
Figure 4 Serum IgG antibody responses at 21 days following primary immunization (m) and reimmunization (m) of mice with graded doses of A/Beijing X109 subunit vaccine
Table 2 Serum IgG antibody response and protection against challenge virus infection following immunization with graded doses of A/Beijing X109 subunit vaccine Vaccine in DTP
Vaccine in saline Dose of A/Beijing x109 subunit vaccine
Serum IgG antibody ELISA reading (mean +s.d.)
No. of infections
(pg HA)
No. of mice tested
3 weeks after 1st immunization
3 weeks after 2nd immunization
Lung
5.0 1.25 0.30 0.08 0.02 Control
7 a 8 a 8 a
0.225f0.02 0.144kO.02 0.118iO.01 0.097f0.06 0.095 + 0.01 0.032 f 0.11
0.656 + 0.02 0.368+0.03 0.360 + 0.01 0.295 kO.02 0.240 kO.01 0.039+0.01
l/7 518 7/a 718 718 718
Serum IgG antibody ELISA reading (mean+s.d.)
No. of infections
Nasal wash
No. of mice tested
3 weeks after 1st immunization
3 weeks after 2nd immunization
Lung
Nasal wash
317
a
718
a
818 818 818 ala
a 8 a 7
1.556*0.08 1.54o_fo.o4 1.480+0.04 0.975 + 0.02 0.844+0.04 0.057f0.08
2.17a*o.ol 1.976t0.04 1.720 & 0.04 1.307f0.02 1.154rtO.06 0.057 + 0.01
018 018 l/8 l/8 118 617
018 tla 118 tla 418 617
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1995 Volume 13 Number 3
257
Response to influenza vaccine in DTP:
C.W. Porter et al.
Table 3 Serum IgG antibody responses and protection against challenge virus infection following immunization with different formulations of A/Beijing X109 subunit vaccine After 1st immunization A/Beijing X109 subunit vaccine (SV) (5 pg HA)
sv + PBS SV + complete DTP SV+alum only SV+DTP less alum Control (PBS)
No. of infections Serum IgG antibody ELISA reading (mean+s.d.)
No. of mice tested
Serum IgG antibody ELISA reading (mean+s.d.)
Lung
Nasal wash
6 8 8 8 8
0.304f0.01 1.316+0.03 0.594*0.01 1.080 + 0.02 0.029 * 0.01
6 8 8 8 8
0.726 k 0.02 1.339 * 0.02 1.041 kO.02 1.498kO.01 0.025iO.01
116 O/8 l/8 O/8 718
216 018 418 O/8 818
1.2 1.0
2; 0.8
52 :Z WE $ J?.
0.6 0.4 0.2 0.0 PBS.
s.v+ PBS
s.v+ ALUM
S.V+DTP less alum
S.V+DTP complete
Figure 5 Serum IgG antibody responses at 21 days after primary immunization (m) and reimmunization (a) with 5pg HA subunit vaccine in alum, subunit vaccine with complete DTP, subunit vaccine with DTP less alum, subunit vaccine with PBS, or PBS alone
DTP was principally due to the presence of the bacterial antigens in the vaccine preparation.
DISCUSSION Although many authorities have indicated the value of immunization against influenza in infants and children with a history of recurrent respiratory tract infections or otitis mediajm5, others have not supported this suggestion for practical reasons ~3,’. Many of the objections could be overcome if immunization against influenza were incorporated into recommended vaccine policies, and the present study investigated one such strategy: (i) the simple mixture of influenza subunit vaccine with DTP vaccine combines two vaccines each shown to be safe for children18; (ii) the reported adjuvant effect of alum and Bordetella pertussis on the immune response could allow smaller quantities of influenza vaccine to be used, thus reducing further the reactogenicity of the vaccine”,‘g; (iii) adjuvants are reported to broaden the immune response which could enhance immunity to influenzazO; and (iv) it would not require a further inoculation and interference with recommended vaccine schedulesg. The present results show that incorporation of
258
Vaccine
(day 42)
No. of mice tested
1.61
g $
After 2nd immunization
(day 21)
1995 Volume
13 Number 3
influenza subunit vaccine into DTP enhanced the serum IgG response, broadened the IgG subclass antibody response, was antigen sparing, and increased the level of protection against challenge virus infection by a factor of 250 or more. Experiments were carried out with complete DTP combined with subunit vaccine dialysed to remove phosphates and allow adsorption of the viral antigens to the alum; however, the results observed (not shown) were similar to those seen for simple mixtures of virus vaccine and DTP, suggesting that the adjuvant effect of DTP is similar to that seen with muramyl dipeptides where physical association between antigen and adjuvant is not a requirement for effective adjuvant action’l. The adjuvant effects of DTP on the immune respol;ses to the influenza subunit vaccine were attributed mainly to the bacterial antigen components of DTP, particularly to the B. pertussis component as whole organisms of this species are known to have adjuvant activitylg. In addition, the stimulation of cell-mediated immune responses by DTP (see below) is probably due to this bacterial component, since alum is a poor or non-inducer of these responses ** . Because of possible reactivity in children to whole-cell B. pertussis, investigators are at present researching subcellular fractions of this organism for use in human vaccines: it is not known whether subcellular fractions would have the same adjuvant activity as whole organisms, and it is therefore suggested that some of the present experiments should be repeated using DTP containing subcellular fractions of B. pertussis. The present studies show that three doses of subunit vaccine in saline produced high levels of serum IgG antibody but failed to give complete protection against challenge virus infections: in contrast, no infections were seen following live virus challenge in mice given three doses of vaccine in DTP. The results suggest that the vaccine in DTP produced a more solid immunity, probably by broadening the immune responses and by induction of immune parameters not induced by saline vaccine. This effect cannot be attributed to production of local antibody, which as been shown to be important in the induction of immunity23324: the present studies indicate that both saline- and DTP-formulated vaccines induced relatively low and similar levels of local IgA antibody (data not shown). Possibly more important is the induction of IgG subclass antibodies: saline vaccine principally induced an IgGl antibody response, whilst vaccine in DTP induced a marked increase in IgG2a and IgG2b antibody. Previous studies have shown that the
Response to influenza
production of TgG2a antibodies is associated with the production of interleukin-2 and interferon-y*‘; these cytokines are secreted by Thi-I subset T lymphocytes26,27 which mediate a range of immune responses, including neutralizing activity**, complement activation29, phagocytosis3’, and cell-mediated immune responses31. Thus, it is possible that the enhanced immunity induced by the subunit vaccine in DTP is due to broader immune responses. The present results have been obtained in experiments in mice and cannot be extrapolated to man: immune reactions to vaccines and adjuvant effects are recognized to differ from species to species3*. The present results were obtained with a different adjuvant system to those used previously which further limits extrapolation. However, the results suggest that incorporation of subunit influenza A vaccine into DTP may have many advantages as listed above, and indicate that studies should now be carried out in human subjects. Apart from the enhanced immunity observed in mice given subunit influenza vaccine in DTP, the formulation was antigen sparing: this offers a potential advantage in time of vaccine shortage and a further reduction in the influenza vaccine reactivity that occurs due to the innate toxicity of the virus haemagglutinin33. Should DTP be shown to enhance the immune response and protection against influenza in humans, the results offer a simple strategy for the immunization of at-risk infants and children to control infection: a wider strategy could limit infection in this age group which is suggested to be a major source of infection for other children and for adults. There is also potential for production of a less reactive influenza vaccine preparation for adults.
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ACKNOWLEDGEMENTS C.W.P. is supported by the Yorkshire Cancer Research Campaign. The authors are grateful for the generous gift of influenza A/Beijing X109 virus, subunit vaccine and DTP vaccine from Dr J. Furminger and Dr T. Colgate, Evans Medical, Speke, Liverpool, UK, and the expert technical assistance of Mrs T. Smith.
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REFERENCES Potter, C.W. Inactivated influenza virus vaccine. In: Basic and Applied Influenza Research (Ed. Beare, AS.), CRC Press, Boca Raton, Florida, 1982, pp. 119-158 Prevention and Control of Influenza. Recommendations for the lmmunization Practices Advisory Committee (ACIP). MMWR 1992, 41, l-17 Glezen. W.P., Paredes, A. and Taber, L.H. Influenza in children. Relationship to other respiratory agents. JAMA 1986,243,1345-1349 Heikkinen, T., Rouskanen, O., Waris, M., Ziegler, T., Arola, M. and Halonen, P. Influenza vaccination in the prevention of acute otitis media in children. Am. J. Dis. Child. 1991, 145, 445-448 Glezen,W.P., Taber, L.H., Gruber, W.C., Piedra, P.A., Clover, R.D., Demmler, R.W. et al. Family studies of vaccine efficacy in children: comparison of protection provided by inactivated and attenuated influenza vaccines. In: Oprions for the Control of Influenza, volume II (Eds Hannoun, C., Kendal, A.P., Klenk, H.D. and Ruben, F.L.) Excerpta Medica, Amsterdam, 1993, pp. 435-437 Glezen, W.P. Considerations of the risk of influenza in children and indications for prophylaxis. Rev. Infect. Dis. 1980, 2, 40
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Hall, C.E., Cooney. M.K. and Fox, J.P. The Seattle virus watch. IV. Comparative epidemiological observations of infections with influenza A and B viruses, 1965-1969, in families with young children. Am. J. fpidemiol. 1973, 98, 366-386 Nicholson, K.G., Tyrrell, D.A.J., Harrison, P., Potter, C.W., Jennings, R., Clark, A. et a/. Clinical studies of monovalent inactivated whole virus and subunit A/USSR/77 (HlNl) vaccine: serological responses and clinical reactions. J. Biol. Stand. 1979, 7, 123-136 Potter, C.W. Vaccinations of infants and children: conclusions and recommendations. In: Options for the Control of Influenza, volume II (Eds Hannoun, C., Kendal, A.P., Klenk, H.D. and Ruben, F.L.) Excerpta Medica, Amsterdam, 1993, pp. 449-456 Hennessy, A.V. and Davenport, F.M. Studies on vaccination of infants against influenza with haemagglutinin. Proc. Sot. Exp. Biol. Med. 1974,146, 206-204 Watemberg, N., Dagan, R., Arbelli, I., Belmaker, I., Morag, A. and Hessel, L. Safety and immunogenicity of Haemophilus type b tetanus protein conjugate vaccine, mixed in the same syringe with diphtheria-tetanus-pertussis vaccine in young infants. Pediatr. Infect. Dis. J. 1991, 10, 758-763 Barra, A., Dagan, R., Preud’homme, J.L., Bajart, A., Danve, B. and Fritzel, B. Characterization of the serum antibody response by Haemophilus influenza type b tetanus protein conjugate vaccine in infants receiving a DTP-combined vaccine from 2 months of age. Vaccine 1993,ll. 1003-1006 Al-Khayatt. R., Jennings, R. and Potter, C.W. Interpretation of responses and protective levels of antibody against attenuated influenza A viruses using single radial haemolysis. J. Hyg. (Camb.) 1984, 92, 301-312 Jennings, R., Smith, T.L., Spencer, R.C., Mellersh, A.M., Edey, D., Fenton, P. et al. Inactivated influenza vaccines in man: a comparative study of subunit and split vaccines using two methods of assessment of antibody responses. Vaccine 1984, 2, 75-86 Ben Ahmeida, E.T., Jennings, R., Erturk, M. and Potter, C.W. The IgA and subclass IgG response and protection in mice immunised with influenza antigens administered as ISCOMs, with FCA. ALH or as infectious virus. Arch. Viral. 1992, 125. 71-86 Potter, C.W. and Oxford, J.S. Determinants of immunity to influenza infections in man. Br. Med. Bull. 1979, 3.5, 69-75 Webster, R.G. and Thomas, T.L. Efficacy of equine influenza vaccines for protection against AIEquinelJilinl89 (H3N8) - a new equine influenza virus. Vaccine 1993, 11, 987-991 Miles, R., Potter, C.W., Clark, A. and Jennings, R. A comparative study of the reactogenicity and immunogenicity of two inactivated influenza vaccines in children. J. Biol. Stand. 1982, 10, 59-68 Dresser, D.W., Works, H.H. and Anderson, H.R. The effect of pertussis vaccine on the immune response of mice to sheep red blood cells. C/in. fxp. Immunol. 1971, 7, 817-831 Halman, C. New approaches to vaccine delivery. In: Opfions for the Control of Influenza, volume II (Eds Hannoun, C., Kendal, A.P., Klenk, H.D. and Ruben, F.L.) Excerpta Medica, Amsterdam, 1993, pp. 389-391 Lecerc, C. and Vogel, F. Synthetic immunomodulators and synthetic vaccines. Crit. Rev. Thera. Drug Carrier Syst. 1986, 2, 353-369 Bomford, R. The comparative selectivity of adjuvants for humoral and cell-mediated immunity. C/in. Exp. Immunol. 1980,39,435-448 Freestone, D.S., Hamilton-Smith, S., Schild, G.C., Buckman, R., Chinn, S. and Tyrrell, D.A.J. Antibody responses and resistance to challenge in volunteers vaccinated with live attenuated, detergent split and oil adjuvant AZ/Hong Kong/68 (H3N2) influenza vaccines. J. Hyg. (Camb.) 1972, 70, 531-543 Clements, M.L., Betts, R.F., Tierney, E.L. and Murphy, B.R. Serum and nasal wash antibodies associated with resistance to experimental challenge with influenza A wild-type virus. J. C/in. Microbial. 1986, 24, 157-160 Snapper, CM. and Paul, W.E. Interferon 7 and B cell stimulatory factor-l reciprocally regulate immunoglobulin isotype control. Science 1987, 2.36, 944-947 Mosmann, T.R., Cherwinksi, H.M., Bond, M.W.. Geidlin, M.A. and Coffman. R.L. Two types of murine helper T cell clone. 1. Definition according to profiles of lymphokine activities and secreted proteins. J. Immunol. 1986,136, 2348-2357 KurtJones, E.A., Hambert, S., O’Hara, J.. Paul, WE. and Abbas, A.K. Heterogeneity of helper/inducer T lymphoctes. 1. Lymphokine production and lymphokine responsiveness. J. Exp. Med. 1987,166, 1774-1787 Hocart. M.J., Mackenzie, J.S. and Stewart, G.A. The immunoglobulin G subclass responses of mice to influenza A virus: the effect of mouse strain, and the neutralising activities of individual protein-
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A-purified subclass antibodies. J. Gen. Viral. 1989, 70, 2434-2448 Spiegelberg. H.L. Biological activities of immunoglobulin of different classes and subclasses. Rev. Immunol. 1974.19, 25%B4 Weiner, E. The ability of IgG subclasses to cause the elimination of targets in vivo and to mediate their destruction by phagocytosis in vitro. In: The human IgG subclass: molecule analysis of structure, function and regulation (Ed. Shakib, F.) Pergamon Press, Oxford, 1990, pp. 135-160 Cher, D.J. and Mosmann, T.R. Two types of helper T cell clone.
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2. Delayed type hypersensitivity is mediated by THl clones. J. Immunol. 1997,138,3@84694 Davenport, F.M. Antigenic enhancement of ether-extracted influenza virus vaccines by AIPO.,. Proc. Sot. Exp. Biol. Med. 1968, 127, 557-590 Pickering, J.M.. Smith, H. and Sweet, C. Influenza virus pyrogenicity: central role of structural orientation of virion components and involvement of viral lipid and glycoproteins. J. Gen. Viral. 1992, 73,1345-1354