- Email: [email protected]
Immunogenicity and protection in mice given inactivated influenza vaccine, MPL, QS-21 or QS-7
International Congress Series 1219 (2001) 999 – 1005
Immunogenicity and protection in mice given inactivated inf luenza vaccine, MPL, QS-21 or QS-7 Philip R. Wyde*, Efrain Guzman, Brian E. Gilbert, Robert B. Couch Department of Molecular Virology and Microbiology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA
Abstract Background: Monophosphoryl lipid A (MPL), QS-21 and QS-7 were evaluated in mice for their ability to increase the immunogenicity and protective efficacy of formalin-inactivated (FI) influenza A/Texas/91 virus vaccine. Freund’s incomplete adjuvant (FIA) was used as a positive control. Methods: Mice were inoculated twice, 28 days apart, either intramuscularly (I.M.) with vaccine mixed with phosphate buffered saline, FIA, MPL or QS21, or intranasally (I.N.) with vaccine containing QS-21 or QS-7. The mice were bled on days 0, 28 and 49 and challenged I.N. on this last day with live virus. Four days later, the lungs from each animal were assessed for influenza virus. All sera were tested for virus-specific neutralizing (Nt), hemagglutination inhibiting (HI) and ELISA antibodies. Studies to account for the mechanism(s) of adjuvant activity have been initiated. Results: FIA, MPL and QS-21 all enhanced the production of virus-specific antibodies and increased protection from pulmonary virus infection following I.M. administration. Maximal adjuvanticity occurred in groups inoculated with ‘‘low’’ doses of vaccine and in groups administered vaccine mixed with QS-21. Both QS adjuvants exhibited significant adjuvant activity following I.N. inoculation. Protection correlated best with levels of virus-specific serum Nt and HI antibodies. Conclusions: The present studies support continued development of adjuvants for inactivated influenza virus vaccines. D 2001 Elsevier Science B.V. All rights reserved. Keywords: Adjuvants; Influenza vaccines; Antibodies; QS-21; MPL
1. Introduction Influenza viruses annually cause significant morbidity and mortality worldwide. Although new and innovative vaccines are being developed, inactivated vaccines remain *
Corresponding author. Tel.: +1-713-798-5255; fax: +1-713-798-6802. E-mail address: [email protected] (P.R. Wyde).
0531-5131/01/$ – see front matter D 2001 Elsevier Science B.V. All rights reserved. PII: S 0 5 3 1 - 5 1 3 1 ( 0 1 ) 0 0 4 11 - 3
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the only major modality for prevention of serious disease in the USA and other countries. Unfortunately, these vaccines generally are less effective in the elderly [1 – 3] than in younger individuals, and may not provide full protection from infection in the latter [4]. New adjuvants are being evaluated as one means to improve the immune responses induced by inactivated vaccines [5,6]. In the present studies, monophosphoryl lipid A (MPL; Ribi Adjuvants), QS-21 (Aquila Biopharmaceuticals), QS-7 (Aquila Biopharmaceuticals) and traditional Freund’s incomplete adjuvant (FIA) were compared in mice for their ability to enhance the immunogenicity and protective efficacy of a commercially produced inactivated monovalent influenza A/Texas/91 (H1N1) virus vaccine. MPL and the two QS compounds have been shown to enhance the immunogenicity of different antigens in clinical trials [7,8]. Freund’s incomplete adjuvant (FIA) was included to provide comparisons to an adjuvant that was extensively utilized in the 1950s and 1960s in humans and that has been shown to significantly enhance antibody responses to inactivated influenza virus vaccines [9– 11].
2. Materials and methods 2.1. Viruses The concentrated inactivated monovalent influenza A/Texas/91 (H1N1) virus vaccine (IVV; Aventis Pasteur Laboratories, Swiftwater, PA), live A/Mississippi/86 (H3N2) influenza virus (used to prime mice) and live A/Texas/91 virus (used to challenge mice) utilized in these experiments were all obtained from the Respiratory Pathogens Research Unit, Baylor College of Medicine (BCM). 2.2. Experimental design Groups of five or seven outbred ICR mice were injected intramuscularly (I.M.) or intranasally (I.N.) with IVV mixed with phosphate buffered saline (PBS), FIA, MPL, QS21 or QS-7. Four weeks later, the animals were revaccinated using the same route, adjuvant and dose they had received initially. The mice were bled from the retro-orbital sinus plexus on Day 0, Day 28 and again on Day 49 just before they were challenged I.N. with approximately 100 median infectious doses of live influenza virus. Four days later, each animal was sacrificed and its lungs were removed, processed and assessed for influenza virus as described previously [12]. All sera were tested for virus-specific neutralizing (Nt), hemagglutinin-inhibiting (HI) and ELISA antibodies using previously described methods [13,14]. 2.3. Statistics Titers were transformed to logs and then tested for normal distribution using Bartlett’s test of homogeneity [15]. A parametric analysis of variances (ANOVA) was used for normally distributed data and a nonparametric ANOVA for non-Guassian data.
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3. Results 3.1. Serum antibody responses in mice vaccinated I.M. In initial studies, IVV was tested in unprimed mice using doses of vaccine ranging from 1 to 10 mg virus hemagglutinin (HA)/dose. In addition, an experiment was performed using doses ranging from 1 to 100 mg HA/dose and mice primed with influenza A/ Mississippi/86 (H3N2) virus 8 weeks prior to administering influenza A/Texas vaccine. Data from these experiments are not shown. However, the findings from them closely mirrored those presented in Figs. 1 (virus-specific serum HI response) and 2 (virus-specific serum ELISA antibody response) obtained from an experiment in which unprimed mice and vaccine doses ranging from 1 to 100 mg/dose were used. As Fig. 1 reveals, the serum antibody responses in these experiments followed a dose – response curve, regardless of whether the vaccine was given with or without an adjuvant. However, antibody levels were consistently greater in groups of mice inoculated with vaccine mixed with an adjuvant than in groups inoculated with vaccine without an adjuvant. Maximal fold enhancement of antibody with adjuvant was consistently observed in mice given lower doses of vaccine and in the groups administered vaccine mixed with QS-21. Nevertheless, MPL and FIA given with 1 or 10-mg doses of IVV also induced significantly increased levels of virus-specific antibodies compared to the vaccine –PBS control group. Serum Nt antibody responses in all experiments were similar to the serum HI antibody responses. However, adjuvant effects were less apparent for the virus-specific serum ELISA antibody titers (data not shown).
Fig. 1. Serum hemagglutination-inhibiting (HI) antibody levels on day 49 (21 days post boost) in mice vaccinated twice, 28 days apart, with formalin-inactivated influenza virus vaccine mixed with phosphate buffered saline (PBS; open bars), Freund’s incomplete adjuvant (FIA; diagonal pattern), monophosphoryl lipid A (MPL; hatched pattern) or QS-21 (thin diagonal pattern). Asterisks indicate the mean titer is significantly greater than the titer obtained for animals given the same dose of vaccine and PBS. A + symbol indicates the mean titer is significantly greater than the titer obtained for sera from the mice given vaccine mixed with FIA or MPL.
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3.2. Pulmonary virus titers after virus challenge Pulmonary virus levels in the same mice shown in Fig. 1, 4 days after virus challenge, are displayed in Fig. 2. Lower virus levels were seen in all of the groups of animals given two 1-mg doses of vaccine HA, but only the group inoculated with vaccine and QS-21 had a significantly lower mean virus titer than was seen in the unvaccinated control groups ( p 0.001). The mean virus titer of this group was also significantly less than the groups given two injections containing 1 mg vaccine HA mixed with either FIA or MPL ( p 0.05). In contrast to these results, all of the groups of mice injected with 10-mg doses of vaccine HA mixed with an adjuvant had significantly reduced pulmonary virus titers compared to the titers seen in the unvaccinated control animals. These reductions were also significantly lower than the mean titer of mice given two 10-mg doses of IVV in PBS. However, they were not significantly different from each other. All of the groups vaccinated twice with 100-mg doses of IVV had similar, marked reductions in virus titers that were significantly lower than the mean titers seen in the unvaccinated control mice. Correlation curves indicated that the protection from pulmonary infection was strongly inversely related to the virus-specific Nt and HI antibody titers in sera (R2 0.9), and less related to levels of virus-specific ELISA antibodies (R2 = 0.6, data not shown). 3.3. Responses in mice vaccinated I.N. With QS compounds The mean virus-specific HI antibody titers measured on day 49 in sera of mice inoculated I.N. with two 1-mg doses of virus HA mixed with PBS, QS-21 (10 mg/dose) or QS-7 (50 mg/dose) 28 days apart are shown in the top portion of Fig. 3. As indicated, virus-specific serum antibody was detected only in the groups given vaccine mixed with an adjuvant. The antibody responses in both of the responding groups were
Fig. 2. Pulmonary virus titers determined on day 53, 4 days after virus challenge of the same mice shown in Fig. 1 and this figure. Symbols for statistically significant differences are as in Fig. 1.
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Fig. 3. (Top panel)Virus-specific serum HI antibody levels on day 49 (21 days post boost) in mice intranasally inoculated twice, 28 days apart, with formalin-inactivated influenza virus vaccine mixed with phosphate buffered saline (left bar), QS-21 (10 mg/vaccination; middle bar) or QS-7 (50 mg/vaccination; right-most bar). Asterisks indicate the mean antibody titer is significantly higher than the mean titer obtained for animals given the same dose of vaccine and PBS. (Bottom panel) Pulmonary virus titers determined on day 53, 4 days after virus challenge, in the same mice shown in the top portion of this figure. Asterisks indicate the mean virus titer is significantly lower than the mean titer obtained for animals given the same dose of vaccine and PBS.
equivalent although five-fold more QS-7 than QS-21 was administered with each vaccination. The mean pulmonary virus titers seen in these animals 4 days after virus challenge are shown in the bottom portion of Fig. 3. As indicated, protection of mice from pulmonary virus infection correlated inversely with their serum HI antibody levels and was only seen in the animals given adjuvant.
4. Discussion The data obtained in these studies clearly show that addition of MPL, QS-21 or QS-7 to influenza vaccine led to increased levels of virus-specific antibodies in the sera of test mice (Fig. 1), and increased protection of these animals from pulmonary infection (Fig. 2) when compared to mice given non adjuvanted vaccine. Serum Nt and HI antibody responses exhibited the well-described pattern of an increase with increasing doses of antigen and after a booster inoculation. Of interest was the finding that each of the adjuvants consistently increased serum antibody responses at all doses including the 100-mg HA dose although the adjuvant effect was less pronounced with increasing dose. Of further
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interest was the fact that QS-21 consistently enhanced responses more than either the FIA or MPL adjuvants. To further understand the mechanism(s) involved in the adjuvanticity seen, we have initiated mechanism studies. These include comparing the avidity, IgG subclasses and virus-specific CTL induced by the different adjuvants, as well as the production of local IgA antibody, particularly in animals inoculated I.N. FIA provided a 4- to 20-fold enhancement of serum HI antibodies among mice and humans when used with the inactivated influenza virus vaccines manufactured in the 1950s and 1960s [9– 11]. Vaccines then were generally less pure and were quantified with HA or chick cell agglutination (CCA) units. Enhancement of the latter magnitude has not been consistently reported in recent clinical studies using new adjuvants. Nevertheless, the results of the present study provide a basis for continuing to try and identify suitable adjuvants for enhancement of immune responses and protection using influenza virus vaccines.
Acknowledgements This work was supported by the Respiratory Pathogens Research Unit, Baylor College of Medicine and National Institutes of Health contract AI 65298.
References [1] E.J. Remarque, Influenza vaccination in elderly people, Exp. Gerontol. 34 (1999) 445 – 452. [2] A.M. Palache, W.E. Beyer, M.J. Sprenger, N. Masurel, S. de Jonge, A. Vardy, et al., Antibody response after influenza immunization with various vaccine doses: a double-blind, placebo-controlled, multi-centre, dose – response study in elderly nursing-home residents and young volunteers, Vaccine 11 (1993) 3 – 9. [3] D.C. Powers, R.B. Belshe, Effect of age on cytotoxic T lymphocyte memory as well as serum and local antibody responses elicited by inactivated influenza virus vaccine, J. Infect. Dis. 167 (1993) 584 – 592. [4] R.B. Couch, W.A. Keitel, T.R. Cate, J.A. Quarles, L.A. Taber, W.P. Glezen, Prevention of influenza virus infections by current inactivated influenza virus vaccines, in: L.E. Brown, A.W. Hampson, R.G. Webster (Eds.), Options for the Control of Influenza III, 1996, pp. 97 – 106. Elsevier, Amsterdam. [5] R.K. Gupta, G.R. Siber, Adjuvants for human vaccines — current status, problems and future prospects, Vaccine 13 (1995) 1263 – 1276. [6] D.T. O’Hagan, Recent advances in vaccine adjuvants for systemic and mucosal administration, J. Pharm. Pharmacol. 49 (1997) 1 – 10. [7] J.R. Baldridge, R.T. Crane, Monophosphoryl lipid A (MPL) formulations for the next generation of vaccines, Methods 19 (1999) 103 – 107. [8] C.R. Kensil, J.Y. Wu, C.A. Anderson, D.A. Wheeler, J. Amsden, QS-21 and QS-7: purified saponin adjuvants, Dev. Biol. Stand. 92 (1998) 41 – 47. [9] A.C. Allison, N.E. Byars, Immunological adjuvants and their mode of action, in: R.W. Ellis (Ed.), Vaccines: New Approaches to Immunologic Problems, Butterworth-Heinemann, Stoneham, MA, 1992, pp. 431 – 449. [10] A.V. Hennessy, F.M. Davenport, Relative merits of aqueous and adjuvant influenza vaccines when used in a two-dose schedule, Public Health Rep. 76 (1961) 411 – 419. [11] J.E. Salk, M.L. Bailey, A.M. Laurent, The use of adjuvants in studies on influenza immunization: II. Increased antibody formation in human subjects inoculated with influenza virus vaccine in a water-in-oil emulsion, Am. J. Hyg. 55 (1952) 439 – 456. [12] P.R. Wyde, B.E. Gilbert, B.M. Levy, Evidence that T-lymphocytes are part of the blood – brain barrier to virus dissemination, J. Neuroimmunol. 5 (1983) 47 – 58.
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[13] R.Q. Robinson, W.R. Dowdle, Influenza viruses, in: E.H. Schmidt, N.J. Lennette (Eds.), Diagnostic Procedures for Viral and Rickettsial Infections, American Public Health Association, New York, NY, 1969, pp. 414 – 433. [14] S.-I. Tamura, Y. Samegai, H. Kurate, T. Nagamine, C. Aizawa, T. Kurata, Protection against influenza virus infection by vaccine inoculated intranasally with cholera toxin B subunit, Vaccine 6 (1988) 409 – 413. [15] R.R. Sokal, F.J. Rohlf, Biometry, Freeman, San Francisco, 1969, p. 370.
Immunogenicity and protection in mice given inactivated inf luenza vaccine, MPL, QS-21 or QS-7 Philip R. Wyde*, Efrain Guzman, Brian E. Gilbert, Robert B. Couch Department of Molecular Virology and Microbiology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA
Abstract Background: Monophosphoryl lipid A (MPL), QS-21 and QS-7 were evaluated in mice for their ability to increase the immunogenicity and protective efficacy of formalin-inactivated (FI) influenza A/Texas/91 virus vaccine. Freund’s incomplete adjuvant (FIA) was used as a positive control. Methods: Mice were inoculated twice, 28 days apart, either intramuscularly (I.M.) with vaccine mixed with phosphate buffered saline, FIA, MPL or QS21, or intranasally (I.N.) with vaccine containing QS-21 or QS-7. The mice were bled on days 0, 28 and 49 and challenged I.N. on this last day with live virus. Four days later, the lungs from each animal were assessed for influenza virus. All sera were tested for virus-specific neutralizing (Nt), hemagglutination inhibiting (HI) and ELISA antibodies. Studies to account for the mechanism(s) of adjuvant activity have been initiated. Results: FIA, MPL and QS-21 all enhanced the production of virus-specific antibodies and increased protection from pulmonary virus infection following I.M. administration. Maximal adjuvanticity occurred in groups inoculated with ‘‘low’’ doses of vaccine and in groups administered vaccine mixed with QS-21. Both QS adjuvants exhibited significant adjuvant activity following I.N. inoculation. Protection correlated best with levels of virus-specific serum Nt and HI antibodies. Conclusions: The present studies support continued development of adjuvants for inactivated influenza virus vaccines. D 2001 Elsevier Science B.V. All rights reserved. Keywords: Adjuvants; Influenza vaccines; Antibodies; QS-21; MPL
1. Introduction Influenza viruses annually cause significant morbidity and mortality worldwide. Although new and innovative vaccines are being developed, inactivated vaccines remain *
Corresponding author. Tel.: +1-713-798-5255; fax: +1-713-798-6802. E-mail address: [email protected] (P.R. Wyde).
0531-5131/01/$ – see front matter D 2001 Elsevier Science B.V. All rights reserved. PII: S 0 5 3 1 - 5 1 3 1 ( 0 1 ) 0 0 4 11 - 3
1000
P.R. Wyde et al. / International Congress Series 1219 (2001) 999–1005
the only major modality for prevention of serious disease in the USA and other countries. Unfortunately, these vaccines generally are less effective in the elderly [1 – 3] than in younger individuals, and may not provide full protection from infection in the latter [4]. New adjuvants are being evaluated as one means to improve the immune responses induced by inactivated vaccines [5,6]. In the present studies, monophosphoryl lipid A (MPL; Ribi Adjuvants), QS-21 (Aquila Biopharmaceuticals), QS-7 (Aquila Biopharmaceuticals) and traditional Freund’s incomplete adjuvant (FIA) were compared in mice for their ability to enhance the immunogenicity and protective efficacy of a commercially produced inactivated monovalent influenza A/Texas/91 (H1N1) virus vaccine. MPL and the two QS compounds have been shown to enhance the immunogenicity of different antigens in clinical trials [7,8]. Freund’s incomplete adjuvant (FIA) was included to provide comparisons to an adjuvant that was extensively utilized in the 1950s and 1960s in humans and that has been shown to significantly enhance antibody responses to inactivated influenza virus vaccines [9– 11].
2. Materials and methods 2.1. Viruses The concentrated inactivated monovalent influenza A/Texas/91 (H1N1) virus vaccine (IVV; Aventis Pasteur Laboratories, Swiftwater, PA), live A/Mississippi/86 (H3N2) influenza virus (used to prime mice) and live A/Texas/91 virus (used to challenge mice) utilized in these experiments were all obtained from the Respiratory Pathogens Research Unit, Baylor College of Medicine (BCM). 2.2. Experimental design Groups of five or seven outbred ICR mice were injected intramuscularly (I.M.) or intranasally (I.N.) with IVV mixed with phosphate buffered saline (PBS), FIA, MPL, QS21 or QS-7. Four weeks later, the animals were revaccinated using the same route, adjuvant and dose they had received initially. The mice were bled from the retro-orbital sinus plexus on Day 0, Day 28 and again on Day 49 just before they were challenged I.N. with approximately 100 median infectious doses of live influenza virus. Four days later, each animal was sacrificed and its lungs were removed, processed and assessed for influenza virus as described previously [12]. All sera were tested for virus-specific neutralizing (Nt), hemagglutinin-inhibiting (HI) and ELISA antibodies using previously described methods [13,14]. 2.3. Statistics Titers were transformed to logs and then tested for normal distribution using Bartlett’s test of homogeneity [15]. A parametric analysis of variances (ANOVA) was used for normally distributed data and a nonparametric ANOVA for non-Guassian data.
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3. Results 3.1. Serum antibody responses in mice vaccinated I.M. In initial studies, IVV was tested in unprimed mice using doses of vaccine ranging from 1 to 10 mg virus hemagglutinin (HA)/dose. In addition, an experiment was performed using doses ranging from 1 to 100 mg HA/dose and mice primed with influenza A/ Mississippi/86 (H3N2) virus 8 weeks prior to administering influenza A/Texas vaccine. Data from these experiments are not shown. However, the findings from them closely mirrored those presented in Figs. 1 (virus-specific serum HI response) and 2 (virus-specific serum ELISA antibody response) obtained from an experiment in which unprimed mice and vaccine doses ranging from 1 to 100 mg/dose were used. As Fig. 1 reveals, the serum antibody responses in these experiments followed a dose – response curve, regardless of whether the vaccine was given with or without an adjuvant. However, antibody levels were consistently greater in groups of mice inoculated with vaccine mixed with an adjuvant than in groups inoculated with vaccine without an adjuvant. Maximal fold enhancement of antibody with adjuvant was consistently observed in mice given lower doses of vaccine and in the groups administered vaccine mixed with QS-21. Nevertheless, MPL and FIA given with 1 or 10-mg doses of IVV also induced significantly increased levels of virus-specific antibodies compared to the vaccine –PBS control group. Serum Nt antibody responses in all experiments were similar to the serum HI antibody responses. However, adjuvant effects were less apparent for the virus-specific serum ELISA antibody titers (data not shown).
Fig. 1. Serum hemagglutination-inhibiting (HI) antibody levels on day 49 (21 days post boost) in mice vaccinated twice, 28 days apart, with formalin-inactivated influenza virus vaccine mixed with phosphate buffered saline (PBS; open bars), Freund’s incomplete adjuvant (FIA; diagonal pattern), monophosphoryl lipid A (MPL; hatched pattern) or QS-21 (thin diagonal pattern). Asterisks indicate the mean titer is significantly greater than the titer obtained for animals given the same dose of vaccine and PBS. A + symbol indicates the mean titer is significantly greater than the titer obtained for sera from the mice given vaccine mixed with FIA or MPL.
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3.2. Pulmonary virus titers after virus challenge Pulmonary virus levels in the same mice shown in Fig. 1, 4 days after virus challenge, are displayed in Fig. 2. Lower virus levels were seen in all of the groups of animals given two 1-mg doses of vaccine HA, but only the group inoculated with vaccine and QS-21 had a significantly lower mean virus titer than was seen in the unvaccinated control groups ( p 0.001). The mean virus titer of this group was also significantly less than the groups given two injections containing 1 mg vaccine HA mixed with either FIA or MPL ( p 0.05). In contrast to these results, all of the groups of mice injected with 10-mg doses of vaccine HA mixed with an adjuvant had significantly reduced pulmonary virus titers compared to the titers seen in the unvaccinated control animals. These reductions were also significantly lower than the mean titer of mice given two 10-mg doses of IVV in PBS. However, they were not significantly different from each other. All of the groups vaccinated twice with 100-mg doses of IVV had similar, marked reductions in virus titers that were significantly lower than the mean titers seen in the unvaccinated control mice. Correlation curves indicated that the protection from pulmonary infection was strongly inversely related to the virus-specific Nt and HI antibody titers in sera (R2 0.9), and less related to levels of virus-specific ELISA antibodies (R2 = 0.6, data not shown). 3.3. Responses in mice vaccinated I.N. With QS compounds The mean virus-specific HI antibody titers measured on day 49 in sera of mice inoculated I.N. with two 1-mg doses of virus HA mixed with PBS, QS-21 (10 mg/dose) or QS-7 (50 mg/dose) 28 days apart are shown in the top portion of Fig. 3. As indicated, virus-specific serum antibody was detected only in the groups given vaccine mixed with an adjuvant. The antibody responses in both of the responding groups were
Fig. 2. Pulmonary virus titers determined on day 53, 4 days after virus challenge of the same mice shown in Fig. 1 and this figure. Symbols for statistically significant differences are as in Fig. 1.
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Fig. 3. (Top panel)Virus-specific serum HI antibody levels on day 49 (21 days post boost) in mice intranasally inoculated twice, 28 days apart, with formalin-inactivated influenza virus vaccine mixed with phosphate buffered saline (left bar), QS-21 (10 mg/vaccination; middle bar) or QS-7 (50 mg/vaccination; right-most bar). Asterisks indicate the mean antibody titer is significantly higher than the mean titer obtained for animals given the same dose of vaccine and PBS. (Bottom panel) Pulmonary virus titers determined on day 53, 4 days after virus challenge, in the same mice shown in the top portion of this figure. Asterisks indicate the mean virus titer is significantly lower than the mean titer obtained for animals given the same dose of vaccine and PBS.
equivalent although five-fold more QS-7 than QS-21 was administered with each vaccination. The mean pulmonary virus titers seen in these animals 4 days after virus challenge are shown in the bottom portion of Fig. 3. As indicated, protection of mice from pulmonary virus infection correlated inversely with their serum HI antibody levels and was only seen in the animals given adjuvant.
4. Discussion The data obtained in these studies clearly show that addition of MPL, QS-21 or QS-7 to influenza vaccine led to increased levels of virus-specific antibodies in the sera of test mice (Fig. 1), and increased protection of these animals from pulmonary infection (Fig. 2) when compared to mice given non adjuvanted vaccine. Serum Nt and HI antibody responses exhibited the well-described pattern of an increase with increasing doses of antigen and after a booster inoculation. Of interest was the finding that each of the adjuvants consistently increased serum antibody responses at all doses including the 100-mg HA dose although the adjuvant effect was less pronounced with increasing dose. Of further
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interest was the fact that QS-21 consistently enhanced responses more than either the FIA or MPL adjuvants. To further understand the mechanism(s) involved in the adjuvanticity seen, we have initiated mechanism studies. These include comparing the avidity, IgG subclasses and virus-specific CTL induced by the different adjuvants, as well as the production of local IgA antibody, particularly in animals inoculated I.N. FIA provided a 4- to 20-fold enhancement of serum HI antibodies among mice and humans when used with the inactivated influenza virus vaccines manufactured in the 1950s and 1960s [9– 11]. Vaccines then were generally less pure and were quantified with HA or chick cell agglutination (CCA) units. Enhancement of the latter magnitude has not been consistently reported in recent clinical studies using new adjuvants. Nevertheless, the results of the present study provide a basis for continuing to try and identify suitable adjuvants for enhancement of immune responses and protection using influenza virus vaccines.
Acknowledgements This work was supported by the Respiratory Pathogens Research Unit, Baylor College of Medicine and National Institutes of Health contract AI 65298.
References [1] E.J. Remarque, Influenza vaccination in elderly people, Exp. Gerontol. 34 (1999) 445 – 452. [2] A.M. Palache, W.E. Beyer, M.J. Sprenger, N. Masurel, S. de Jonge, A. Vardy, et al., Antibody response after influenza immunization with various vaccine doses: a double-blind, placebo-controlled, multi-centre, dose – response study in elderly nursing-home residents and young volunteers, Vaccine 11 (1993) 3 – 9. [3] D.C. Powers, R.B. Belshe, Effect of age on cytotoxic T lymphocyte memory as well as serum and local antibody responses elicited by inactivated influenza virus vaccine, J. Infect. Dis. 167 (1993) 584 – 592. [4] R.B. Couch, W.A. Keitel, T.R. Cate, J.A. Quarles, L.A. Taber, W.P. Glezen, Prevention of influenza virus infections by current inactivated influenza virus vaccines, in: L.E. Brown, A.W. Hampson, R.G. Webster (Eds.), Options for the Control of Influenza III, 1996, pp. 97 – 106. Elsevier, Amsterdam. [5] R.K. Gupta, G.R. Siber, Adjuvants for human vaccines — current status, problems and future prospects, Vaccine 13 (1995) 1263 – 1276. [6] D.T. O’Hagan, Recent advances in vaccine adjuvants for systemic and mucosal administration, J. Pharm. Pharmacol. 49 (1997) 1 – 10. [7] J.R. Baldridge, R.T. Crane, Monophosphoryl lipid A (MPL) formulations for the next generation of vaccines, Methods 19 (1999) 103 – 107. [8] C.R. Kensil, J.Y. Wu, C.A. Anderson, D.A. Wheeler, J. Amsden, QS-21 and QS-7: purified saponin adjuvants, Dev. Biol. Stand. 92 (1998) 41 – 47. [9] A.C. Allison, N.E. Byars, Immunological adjuvants and their mode of action, in: R.W. Ellis (Ed.), Vaccines: New Approaches to Immunologic Problems, Butterworth-Heinemann, Stoneham, MA, 1992, pp. 431 – 449. [10] A.V. Hennessy, F.M. Davenport, Relative merits of aqueous and adjuvant influenza vaccines when used in a two-dose schedule, Public Health Rep. 76 (1961) 411 – 419. [11] J.E. Salk, M.L. Bailey, A.M. Laurent, The use of adjuvants in studies on influenza immunization: II. Increased antibody formation in human subjects inoculated with influenza virus vaccine in a water-in-oil emulsion, Am. J. Hyg. 55 (1952) 439 – 456. [12] P.R. Wyde, B.E. Gilbert, B.M. Levy, Evidence that T-lymphocytes are part of the blood – brain barrier to virus dissemination, J. Neuroimmunol. 5 (1983) 47 – 58.
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[13] R.Q. Robinson, W.R. Dowdle, Influenza viruses, in: E.H. Schmidt, N.J. Lennette (Eds.), Diagnostic Procedures for Viral and Rickettsial Infections, American Public Health Association, New York, NY, 1969, pp. 414 – 433. [14] S.-I. Tamura, Y. Samegai, H. Kurate, T. Nagamine, C. Aizawa, T. Kurata, Protection against influenza virus infection by vaccine inoculated intranasally with cholera toxin B subunit, Vaccine 6 (1988) 409 – 413. [15] R.R. Sokal, F.J. Rohlf, Biometry, Freeman, San Francisco, 1969, p. 370.