THE JOURNAL OF INFECTIOUS DISEASES. VOL. 135. NO.3. MARCH 1977 © 1977 by the University of Chicago. All rights reserved.
Dose-Response Relationship after Immunization of Volunteers with a New, Surface-Antigen-Adsorbed Influenza Virus Vaccine C. W. Potter, R. Jennings, J. P. Phair, A. Clarke, and C. H. Stuart-Harris
From the Academic Division of Pathology, Department of Virology, University of Sheffield Medical School, Sheffield, Yorkshire, England
The serum antibody response to immunization with different subunit or split-influenza-virus vaccines has been reported to differ considerably. Purified virus hemagglutinin prepared by bromelain treatment [I], vaccines for which virus was disrupted by ether or Tween-80 [2], and deoxycholate-split-virus vaccine have been reported to induce lower titers of serum antibody than whole, inactivated influenza virus vaccines [3, 4]. However, a second virus hemagglutinin preparation and a vaccine for which virus was split by tri- (N-butyl) phosphate were reported to induce titers of antibody comparable to those produced by whole-virus vaccine [5, 6]. Since adverse reactions to immunization are less common after inoculation with split or subunit vaccines than with whole-virus vaccines [6, 7], attempts have been made to increase the immunogenicity of the split-virus vaccines while retaining the propReceived for publication May 11, form July 28, 1976. Please address requests for reprints Department of Virology, University School, Beech Hill Road, Sheffield England.
1976, and in revised to Prof. C. W. Potter, of Sheffield Medical SIO 2RX, Yorkshire,
423
erty of low reactogenicity [8-10]. A surface-antigen-adsorbed vaccine, composed of isolated virus hemagglutinin and neuraminidase adsorbed to an alhydrogel carrier, was found to induce relatively high titers of serum antibody in both animals [11] and volunteers [12]. Furthermore, this vaccine induced higher titers of serum antibody and better immunity to challenge infection in animals than did whole-virus vaccine in saline [11]. The present study reports the dose-response relationship for the serum antibody response to immunization with surface-antigen-adsorbed vaccine prepared from influenza virus A/Port Chalmersj Zd. The production of both homologous HAl and neuraminidase-inhibiting (NI) antibody by different concentrations of vaccine was measured in volunteers. In addition, the production of heterologous and heterotypic serum HAl antibody and of homologous local neutralizing antibody was also measured. Four weeks after immunization, the immunity of all of the volunteers to challenge infection was determined by inoculation of subjects with influenza virus WRL-I05, an attenuated virus closely related
Downloaded from http://jid.oxfordjournals.org/ at University of Birmingham on August 27, 2015
Volunteers (15 per group) were given inoculations of various .doses (5-400 international units [IU]) of surface-antigen-adsorbed influenza virus A/Port Chalmers/ 73 vaccine; this vaccine was prepared from purified virus hemagglutinin and neuraminidase antigens and adsorbed to alhydrogel. The titers of hemagglutinationinhibiting (HAl) and neuraminidase-inhibiting antibodies in serum after immunization showed a clear dose-response relationship. Thus, for volunteers given 400, 100, 25, or 5.0 IV of vaccine, the titers of HAl antibody in serum increased 174-, 32-, 12-, and eightfold, respectively. A similar dose response was observed for production of local neutralizing antibody. Immunization with A/Port Chalmers/73 virus vaccine also induced serum HAl antibody to influenza viruses A/Scotland/74, A/England/72, and A/Hong Kong/68; the increase in titers of antibody to these viruses corresponded directly to the degree of cross-reactivity between the virus tested and the vaccine virus. Four weeks after immunization, all volunteers were challenged with attenuated WRL-I05 influenza virus. Evidence of viral infection was found in one, two, and two volunteers in each group of IS subjects previously immunized with 400, 100, and 25 IV of vaccine, respectively; in six of the IS volunteers given 5.0 IV of vaccine; and in lOaf 15 control subjects. The results suggest that equivalent immunity was induced in volunteers given ~25 IV of vaccine.
Potter et al.
424
antigenically to the surface-antigen-adsorbed vaccine. Materials and Methods
Table 1. Serum antibody response of volunteers to various concentrations of surface-antigen-adsorbed influenza A/Port Chalmers/73 virus vaccine. HAl antibody
NI antibody
Dose of vaccine (IV)
GMT before immunization
GMT after immunization (fold increase in titer)
1:80 15 14 7 4 0
GMT before immunization
5.8 6.6 9.5 15.9 6.5
NOTE. Fifteen volunteers received each dose of vaccine. Titers are reciprocals except where noted. IV GMT = geometric mean titer. Nl = neuraminidase-inhibiting.
GMT after immunization (fold increase in titer)
36.1 21.6 26.0 32.9 5.9
(6.2) (3.3) (2.7) (2.1) (0.9)
= international units.
Downloaded from http://jid.oxfordjournals.org/ at University of Birmingham on August 27, 2015
Virus and virus vaccines. Influenza viruses A/Scotland/74 (H3N2), A/Port Chalmers/Zf (H3N2), A/England/72 (H3N2), A/Hong Kong/68 (H3~2), and A/Singapore/57 (H2N2) were obtained from Dr. G. C. Schild, National Institute of Medical Research, Mill Hill, London, England. Pools of virus were prepared from the allantoic fluids of 10-day-old embryonated eggs inoculated with 0.2 ml of a 10-2 .0 dilution of seed virus. After incubation for 48 hr at 33 C, the allantoic fluids were harvested and stored at -80 C. Influenza virus WRL-I05 (H3N2), a recombinant of influenza viruses A/Finland/74 (H3N2) and A/Okuda/57 (H2N2), was supplied by Dr. A. S. Beare, Common Cold Research Centre, Harvard Hospital, Salisbury, England; the properties of this attenuated vaccine virus have been described previously [13]. Immediately before use, the virus was thawed from -80 C and diluted in phosphate-buffered saline (PBS), pH 7.4, to contain 107 . 0 50% egg infectious doses (EID50)/ 0.5 ml. A volume of 0.5 ml of virus was administered to each volunteer intranasally as previously described [12]; the diluted virus was maintained at 4 C and used within I hr of preparation. Surface-antigen-adsorbed influenza virus vaccine was prepared from influenza virus A/Port Chalmers/73 (H3N2). Briefly, egg-grown virus was purified by zonal centrifugation, standard-
ized by international units (IV) [14], and treated with Triton NIOI to release the hemagglutinin and neuraminidase antigens; these antigens were then separated from other viral proteins [9]. A series of surface-antigen-adsorbed vaccines containing 400, 100, 25, or 5.0 IV of viral antigens was prepared by dilution of the purified subunits, and each vaccine was adsorbed to 2.5 mg of alhydrogel in a volume of 0.5 ml. A control vaccine of 2.5 mg of alhydrogel/0.5 ml without viral antigens was also prepared. Experimental design. A group of medical and dental students volunteered for the study; all were healthy and had no known allergy to eggs or to egg protein. A sam ple of serum was taken from each volunteer and tested for HAl antibody to influenza virus A/Port Chalmers/73. From these results, volunteers with HAl antibody titers of > 1:40 were excluded, and the remaining 75 volunteers were divided into five matched groups. Each group contained the same number of volunteers with and without demonstrable HAl antibody in serum, and the mean titer of antibody for the five groups was very similar (table 1). In the assignment of volunteers to groups, no account was taken of titers of NI antibody or of HAl antibody to other influenza A viruses before immunization. Each volunteer in group I was given an im inoculation of 400 IV of surface-antigen-adsorbed A/Port Chalmers/73 virus vaccine in a volume of 0.5 ml; groups 2, 3, 4, and 5 received 100, 25, or 5.0 IV, or alhydrogel alone, respectively. Two nasal washings were obtained from each volunteer in the week before immunization, and additional specimens were collected two and three
Surface-Antigen-Adsorbed Influenza Vaccine
automated method [18], based on the standard technique of the World Health Organization [19]. Nasal washings were tested for neutralizing antibody by the allantois-on-shell method [20]. Shell fragments from four different eggs were used for each dilution of nasal washing, and the titers of neutralizing antibody were calculated by the method of Reed and Muench [21]. Results
Serum antibody response to immunization with surface-antigen-adsorbed A / Port Chalmers/7] virus vaccine. The serum HAl and NI antibody responses of volunteers to im immunization with 400 IV of surface-antigen-adsorbed A/Port Chalmers /Zf virus vaccine are shown in table 2. Before immunization, low levels of serum HAl antibody were detected in only five volunteers in this group (no. 11-15), and immunization induced serum HAl antibody to levels of ~1 :640 in all 15 volunteers. The geometric mean titer (GMT) of HAl antibody in serum increased from 1:7.6 to 1: 1,325.4 for volunteers given 400 IV of vaccine. For the 10 subjects with no detectable HAl antibody (titers of 1:80 have been reported to be protective and titers of < 1:40 are probably not [22]. All 15 volunteers given 400 IV of vaccine developed serum HAl antibody titers of > 1:80, whereas volunteers given alhydrogel alone showed no increase in titer of antibody in serum. For subjects given smaller doses of vac-
Downloaded from http://jid.oxfordjournals.org/ at University of Birmingham on August 27, 2015
weeks after immunization; these washings were taken and processed for antibody determination as described previously [15]. Three weeks after immunization, a second specimen of blood was collected from each volunteer. Four weeks after immunization with surfaceantigen-adsorbed vaccine, each volunteer was given an intranasal inoculation of attenuated influenza virus WRL-I05; the conditions of viral inoculation were strictly controlled since variation in technique can alter the number of infections significantly. The virus in 0.5 ml of PBS was given drop-wise into the nostrils of subjects lying on an examination couch with the head extended over the edge. After inoculation, volunteers remained lying for an additional 2 min and on rising were asked not to blow their noses for an additional 1 hr. Throat swabs, which were placed in medium 199 (Gibco-Biocult., Paisley, Scotland) containing 2.0% bovine serum albumin and antibiotics, and nasal washings for viral isolation were obtained three days after viral inoculation, and an additional blood sample was taken 18 days later. Isolation of virus or a significant rise in HAl and/or CF antibody was taken as evidence of infection with the challenge virus. Isolation of ViTUS. Nasal washings and throat swabs for viral isolation were stored at -80 C prior to testing. After thawing, each specimen was inoculated by the allantoic route into l.O-dayold embryonated eggs. The eggs were incubated at 33 C for 48 hr, after which the allantoic fluids were harvested and tested for virus by hemagglutination with fowl cells. All viral isolates were identified by HAl tests with use of monospecific ferret antisera. Serological tests. HAl tests were carried out by a modification of the microtiter method [16] as described previously [12]. CF tests were performed by the method of Bradstreet and Taylor [17] with use of 2.5 50% minimal hemagglutinating doses of guinea pig com plement and A / Port Chalmers/73 virus soluble antigen extracted from the chorioallantoic membranes of virus-infected eggs. The mixtures were incubated overnight at 4 C before addition of sensitized sheep erythrocytes, and the titer was taken as the highest dilution of serum that gave ~75% fixation of complement. Tests for NI antibody were performed by the
425
Potter et al.
426
Table 2. Serum antibody response to immunization with 400 international units of surface-antigen-adsorbed influenza A/Port Chalmersf7 3 virus vaccine. HAl antibody Volunteer no.
Before immunization
Dose-Response Relationship after Immunization of Volunteers with a New, Surface-Antigen-Adsorbed Influenza Virus Vaccine C. W. Potter, R. Jennings, J. P. Phair, A. Clarke, and C. H. Stuart-Harris
From the Academic Division of Pathology, Department of Virology, University of Sheffield Medical School, Sheffield, Yorkshire, England
The serum antibody response to immunization with different subunit or split-influenza-virus vaccines has been reported to differ considerably. Purified virus hemagglutinin prepared by bromelain treatment [I], vaccines for which virus was disrupted by ether or Tween-80 [2], and deoxycholate-split-virus vaccine have been reported to induce lower titers of serum antibody than whole, inactivated influenza virus vaccines [3, 4]. However, a second virus hemagglutinin preparation and a vaccine for which virus was split by tri- (N-butyl) phosphate were reported to induce titers of antibody comparable to those produced by whole-virus vaccine [5, 6]. Since adverse reactions to immunization are less common after inoculation with split or subunit vaccines than with whole-virus vaccines [6, 7], attempts have been made to increase the immunogenicity of the split-virus vaccines while retaining the propReceived for publication May 11, form July 28, 1976. Please address requests for reprints Department of Virology, University School, Beech Hill Road, Sheffield England.
1976, and in revised to Prof. C. W. Potter, of Sheffield Medical SIO 2RX, Yorkshire,
423
erty of low reactogenicity [8-10]. A surface-antigen-adsorbed vaccine, composed of isolated virus hemagglutinin and neuraminidase adsorbed to an alhydrogel carrier, was found to induce relatively high titers of serum antibody in both animals [11] and volunteers [12]. Furthermore, this vaccine induced higher titers of serum antibody and better immunity to challenge infection in animals than did whole-virus vaccine in saline [11]. The present study reports the dose-response relationship for the serum antibody response to immunization with surface-antigen-adsorbed vaccine prepared from influenza virus A/Port Chalmersj Zd. The production of both homologous HAl and neuraminidase-inhibiting (NI) antibody by different concentrations of vaccine was measured in volunteers. In addition, the production of heterologous and heterotypic serum HAl antibody and of homologous local neutralizing antibody was also measured. Four weeks after immunization, the immunity of all of the volunteers to challenge infection was determined by inoculation of subjects with influenza virus WRL-I05, an attenuated virus closely related
Downloaded from http://jid.oxfordjournals.org/ at University of Birmingham on August 27, 2015
Volunteers (15 per group) were given inoculations of various .doses (5-400 international units [IU]) of surface-antigen-adsorbed influenza virus A/Port Chalmers/ 73 vaccine; this vaccine was prepared from purified virus hemagglutinin and neuraminidase antigens and adsorbed to alhydrogel. The titers of hemagglutinationinhibiting (HAl) and neuraminidase-inhibiting antibodies in serum after immunization showed a clear dose-response relationship. Thus, for volunteers given 400, 100, 25, or 5.0 IV of vaccine, the titers of HAl antibody in serum increased 174-, 32-, 12-, and eightfold, respectively. A similar dose response was observed for production of local neutralizing antibody. Immunization with A/Port Chalmers/73 virus vaccine also induced serum HAl antibody to influenza viruses A/Scotland/74, A/England/72, and A/Hong Kong/68; the increase in titers of antibody to these viruses corresponded directly to the degree of cross-reactivity between the virus tested and the vaccine virus. Four weeks after immunization, all volunteers were challenged with attenuated WRL-I05 influenza virus. Evidence of viral infection was found in one, two, and two volunteers in each group of IS subjects previously immunized with 400, 100, and 25 IV of vaccine, respectively; in six of the IS volunteers given 5.0 IV of vaccine; and in lOaf 15 control subjects. The results suggest that equivalent immunity was induced in volunteers given ~25 IV of vaccine.
Potter et al.
424
antigenically to the surface-antigen-adsorbed vaccine. Materials and Methods
Table 1. Serum antibody response of volunteers to various concentrations of surface-antigen-adsorbed influenza A/Port Chalmers/73 virus vaccine. HAl antibody
NI antibody
Dose of vaccine (IV)
GMT before immunization
GMT after immunization (fold increase in titer)
1:80 15 14 7 4 0
GMT before immunization
5.8 6.6 9.5 15.9 6.5
NOTE. Fifteen volunteers received each dose of vaccine. Titers are reciprocals except where noted. IV GMT = geometric mean titer. Nl = neuraminidase-inhibiting.
GMT after immunization (fold increase in titer)
36.1 21.6 26.0 32.9 5.9
(6.2) (3.3) (2.7) (2.1) (0.9)
= international units.
Downloaded from http://jid.oxfordjournals.org/ at University of Birmingham on August 27, 2015
Virus and virus vaccines. Influenza viruses A/Scotland/74 (H3N2), A/Port Chalmers/Zf (H3N2), A/England/72 (H3N2), A/Hong Kong/68 (H3~2), and A/Singapore/57 (H2N2) were obtained from Dr. G. C. Schild, National Institute of Medical Research, Mill Hill, London, England. Pools of virus were prepared from the allantoic fluids of 10-day-old embryonated eggs inoculated with 0.2 ml of a 10-2 .0 dilution of seed virus. After incubation for 48 hr at 33 C, the allantoic fluids were harvested and stored at -80 C. Influenza virus WRL-I05 (H3N2), a recombinant of influenza viruses A/Finland/74 (H3N2) and A/Okuda/57 (H2N2), was supplied by Dr. A. S. Beare, Common Cold Research Centre, Harvard Hospital, Salisbury, England; the properties of this attenuated vaccine virus have been described previously [13]. Immediately before use, the virus was thawed from -80 C and diluted in phosphate-buffered saline (PBS), pH 7.4, to contain 107 . 0 50% egg infectious doses (EID50)/ 0.5 ml. A volume of 0.5 ml of virus was administered to each volunteer intranasally as previously described [12]; the diluted virus was maintained at 4 C and used within I hr of preparation. Surface-antigen-adsorbed influenza virus vaccine was prepared from influenza virus A/Port Chalmers/73 (H3N2). Briefly, egg-grown virus was purified by zonal centrifugation, standard-
ized by international units (IV) [14], and treated with Triton NIOI to release the hemagglutinin and neuraminidase antigens; these antigens were then separated from other viral proteins [9]. A series of surface-antigen-adsorbed vaccines containing 400, 100, 25, or 5.0 IV of viral antigens was prepared by dilution of the purified subunits, and each vaccine was adsorbed to 2.5 mg of alhydrogel in a volume of 0.5 ml. A control vaccine of 2.5 mg of alhydrogel/0.5 ml without viral antigens was also prepared. Experimental design. A group of medical and dental students volunteered for the study; all were healthy and had no known allergy to eggs or to egg protein. A sam ple of serum was taken from each volunteer and tested for HAl antibody to influenza virus A/Port Chalmers/73. From these results, volunteers with HAl antibody titers of > 1:40 were excluded, and the remaining 75 volunteers were divided into five matched groups. Each group contained the same number of volunteers with and without demonstrable HAl antibody in serum, and the mean titer of antibody for the five groups was very similar (table 1). In the assignment of volunteers to groups, no account was taken of titers of NI antibody or of HAl antibody to other influenza A viruses before immunization. Each volunteer in group I was given an im inoculation of 400 IV of surface-antigen-adsorbed A/Port Chalmers/73 virus vaccine in a volume of 0.5 ml; groups 2, 3, 4, and 5 received 100, 25, or 5.0 IV, or alhydrogel alone, respectively. Two nasal washings were obtained from each volunteer in the week before immunization, and additional specimens were collected two and three
Surface-Antigen-Adsorbed Influenza Vaccine
automated method [18], based on the standard technique of the World Health Organization [19]. Nasal washings were tested for neutralizing antibody by the allantois-on-shell method [20]. Shell fragments from four different eggs were used for each dilution of nasal washing, and the titers of neutralizing antibody were calculated by the method of Reed and Muench [21]. Results
Serum antibody response to immunization with surface-antigen-adsorbed A / Port Chalmers/7] virus vaccine. The serum HAl and NI antibody responses of volunteers to im immunization with 400 IV of surface-antigen-adsorbed A/Port Chalmers /Zf virus vaccine are shown in table 2. Before immunization, low levels of serum HAl antibody were detected in only five volunteers in this group (no. 11-15), and immunization induced serum HAl antibody to levels of ~1 :640 in all 15 volunteers. The geometric mean titer (GMT) of HAl antibody in serum increased from 1:7.6 to 1: 1,325.4 for volunteers given 400 IV of vaccine. For the 10 subjects with no detectable HAl antibody (titers of 1:80 have been reported to be protective and titers of < 1:40 are probably not [22]. All 15 volunteers given 400 IV of vaccine developed serum HAl antibody titers of > 1:80, whereas volunteers given alhydrogel alone showed no increase in titer of antibody in serum. For subjects given smaller doses of vac-
Downloaded from http://jid.oxfordjournals.org/ at University of Birmingham on August 27, 2015
weeks after immunization; these washings were taken and processed for antibody determination as described previously [15]. Three weeks after immunization, a second specimen of blood was collected from each volunteer. Four weeks after immunization with surfaceantigen-adsorbed vaccine, each volunteer was given an intranasal inoculation of attenuated influenza virus WRL-I05; the conditions of viral inoculation were strictly controlled since variation in technique can alter the number of infections significantly. The virus in 0.5 ml of PBS was given drop-wise into the nostrils of subjects lying on an examination couch with the head extended over the edge. After inoculation, volunteers remained lying for an additional 2 min and on rising were asked not to blow their noses for an additional 1 hr. Throat swabs, which were placed in medium 199 (Gibco-Biocult., Paisley, Scotland) containing 2.0% bovine serum albumin and antibiotics, and nasal washings for viral isolation were obtained three days after viral inoculation, and an additional blood sample was taken 18 days later. Isolation of virus or a significant rise in HAl and/or CF antibody was taken as evidence of infection with the challenge virus. Isolation of ViTUS. Nasal washings and throat swabs for viral isolation were stored at -80 C prior to testing. After thawing, each specimen was inoculated by the allantoic route into l.O-dayold embryonated eggs. The eggs were incubated at 33 C for 48 hr, after which the allantoic fluids were harvested and tested for virus by hemagglutination with fowl cells. All viral isolates were identified by HAl tests with use of monospecific ferret antisera. Serological tests. HAl tests were carried out by a modification of the microtiter method [16] as described previously [12]. CF tests were performed by the method of Bradstreet and Taylor [17] with use of 2.5 50% minimal hemagglutinating doses of guinea pig com plement and A / Port Chalmers/73 virus soluble antigen extracted from the chorioallantoic membranes of virus-infected eggs. The mixtures were incubated overnight at 4 C before addition of sensitized sheep erythrocytes, and the titer was taken as the highest dilution of serum that gave ~75% fixation of complement. Tests for NI antibody were performed by the
425
Potter et al.
426
Table 2. Serum antibody response to immunization with 400 international units of surface-antigen-adsorbed influenza A/Port Chalmersf7 3 virus vaccine. HAl antibody Volunteer no.
Before immunization
