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Intranasal immunization with inactivated influenza vaccine
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PSTT Vol. 2, No. 10 October 1999
Intranasal immunization with inactivated influenza vaccine Chris W. Potter and Roy Jennings The development of improved vaccines against epidemic and pandemic influenza virus infection remains a priority in vaccine research. Killed vaccines given by injection are both cost-effective and induce immunity; however, their limitations are well known. Live vaccines have been in development for many years, but difficulties and safety concerns have prohibited their licensing in Western countries. However, the newer technologies of vaccine development, including DNA vaccines and attenuated virus vaccines produced by reverse
Many of the problems of vaccine development relate to the antigenic variability of the virus: a rapid mutation rate means that the immune responses to infection or immunization in one year do not necessarily protect against infection in a subsequent year4. Even with this major reservation, the available vaccines are not ideal, and this has led to a search for new strategies. It is with these strategies, and in particular intranasal immunization with inactivated vaccine, that the present review is concerned.
genetics, remain a hope for the future. With these problems in mind, emphasis has been given to the development of inactivated vaccines that are administered intranasally, either as repeated doses of saline vaccine or in conjunction with suitable carriers or adjuvants. This review describes these latter developments and concludes that this approach offers advantages and should be vigorously researched.
Chris W. Potter* and Roy Jennings Sheffield Institute for Vaccine Studies Division of Molecular and Genetic Medicine and Division of Child Health University of Sheffield Medical School Beech Hill Road Sheffield, UK S10 2RX *tel: 144 114 272 4072 fax: 144 114 273 9926 e-mail: [email protected]
402
▼ Data from the past 100 years record the oc-
currence of influenza in most countries in some years, and in some countries in most years.These outbreaks can be local or more widespread, and at intervals of 10–40 years a new virus subtype emerges from a focal point to spread quickly throughout the world to infect most people1. Influenza epidemics remain unpredictable in both time and severity, and the importance of the infection has been recognized for centuries. The virus was first isolated in the laboratory in 1933 (Ref. 2). Vaccines have been developed for more than 60 years3, and vast resources of money and manpower have been devoted to the study of this disease, including the efforts of researchers, physicians, diagnostic laboratories, epidemiologists, pharmaceutical companies and health authorities. Despite this, success in controlling influenza has been limited, and annual outbreaks resulting in numerous deaths continue to occur.
Immunity to influenza The antigens of the influenza virus particles that induce immunity can be identified through using conventional methods. Influenza viruses A and B contain eight RNA fragments, coding for several structural proteins with molecular weights of 10,000–90,000, that make up the virus particles; further proteins, designated nonstructural proteins, are synthesized in infectious cells, but are not present in the completed virions5. These proteins, and the immune responses directed by them, can be studied individually, and, although a response to many of them can be detected, it is the immune response to the surface glycoproteins – haemagglutinin (HA) and neuraminidase (NA) – that constitute the immune state. It follows that all influenza vaccines must contain a relevant HA, and preferably a relevant NA, in the form of whole virus, split virus, subunit virus, live virus or DNA, for immunity to result6. Cell-mediated immune (CMI) responses, including cytotoxic CD81 T lymphocytes that recognize the virus nucleoprotein expressed on the surface of cells containing replicating virus to effect cell lysis7, are transient, but may contribute to immunity, limit clinical infection and provide a broad, heterotypic immunity in the short-term7,8. Many studies have been directed towards identifying the basis of immunity to influenza.
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Unequivocal evidence indicates that a specific serum antibody to the virus HA, haemagglutination-inhibiting (HI) antibody, is the most important immune response, providing immunity to subsequent infection.Thus, susceptibility to challenge virus infection is inversely correlated to the serum HI antibody titre6,9, and passive transfer of HI antibody protects normal and immunodepressed mice from challenge virus infection10.The importance of serum antibody to NA, termed NI antibody, was demonstrated in 1968 when a new virus subtype caused a pandemic; this virus shared the same NA as the virus strains that had caused epidemics in the previous years, but possessed a completely novel HA. Thus, there were individuals at the time who possessed NI antibody but not HI antibody against the new virus, and infection was less common in these subjects than in those without NI antibody, and the higher the titre of serum NI antibody the less common was the infection11. Pure NA vaccine has been shown to induce immunity to clinical disease, but not to infection12. Studies on subjects with varying titres of local IgA antibody and similar titres of serum HI antibody indicate that those with nasal IgA are relatively immune to infection compared with those without this antibody, and the greater the titre of IgA antibody the more solid the immunity13. In addition, animals given anti-IgA lose immunity to challenge virus infection14. The place of CMI in protection remains contentious.There is clear evidence that a CD41 T-lymphocyte response is required for antibody production.This is supported by numerous experiments in naive animals that show that serum antibody does not develop in response to inoculation with a single dose of inactivated virus vaccine, which is a poor inducer of CD41 T cells. Cytotoxic CD81 T lymphocytes do develop in response to inactivated vaccine in primed animals or volunteers and following live virus infection, but this response is relatively short-lived15, although it can be recalled rapidly following later exposure to virus antigen16. The consensus is that CMI responses are required for antibody production and might be important in short-term immunity after viral infection; however, the most important contribution of CMI is to provide a mechanism for limiting the duration of disease. The evidence outlined above indicates that an influenza vaccine should contain the specific HA and NA to stimulate a serum HI antibody response, and this is the prime requirement. Immunity is further strengthened by the presence of serum NI antibody, local IgA antibody and, in the shorter term, by specific CD81 cytotoxic T cells. Inactivated vaccines Inactivated influenza vaccines have been used to induce immunity in volunteers for more than 60 years3. Many of the earlier vaccines were reactogenic, and this experience has left an unfortunate legacy; however, since 1960, when methods to purify
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virus particles became available, these vaccines have been well tolerated. The current inactivated vaccines are produced from a reassortant virus of the current infecting influenza strain and a strain that grows well in embryonated eggs, yielding a virus strain that contains HA and NA of the wild-type virus and which can grow to high titre. The virus is grown in embryonated eggs, harvested, purified by zonal centrifugation and inactivated with either formalin or b-propiolactone. The virus is then used either as a whole virus vaccine, a split virus vaccine in which the intact particles are broken down by chemical means, or a subunit vaccine in which the virus particles are disrupted and only the purified HA and NA form the vaccine17. In each case, the vaccine is standardized to contain approximately 15 mg of HA per dose and is given as a single inoculation, particularly to ‘at-risk’ subjects, which include the elderly, patients with chronic lung or heart disease and those with metabolic disorders18. Because influenza viruses mutate rapidly, the antigenic form in an epidemic virus must be anticipated months in advance to allow time for vaccine production and distribution; modern vaccines contain three virus strains, each at 15 mg of HA per dose, to cover expected infection. At-risk patients, despite earlier concerns that repeated annual injections are not advisable, should receive vaccine annually in anticipation of winter epidemics19. Numerous studies have shown that vaccine induces serum HI antibody to titres above 40 in the majority of vaccinees17; this titre is the reciprocal of the homologous virus antibody titre established by the HI test and is equivalent to a 50% level of protection against challenge virus infection6. Some vaccinees asked to report reactions to vaccine record local redness and soreness; however, these reactions are mild and do not persist beyond 24–48 h, and systemic reactions are rare17. Numerous studies, including both open and closed studies, in which immunized volunteers are given a challenge virus infection, indicate that inactivated influenza vaccines induce immunity in 60–90% of subjects20. One of the main reasons for these relatively disappointing results is the need for vaccine formulation at least five to six months in advance of the influenza season; continuous virus mutation, termed antigenic shift and drift, can result in an epidemic being caused by a virus variant that is distinct from the vaccine strain. In addition, inactivated vaccine given parenterally is a poor inducer of local immunity, which is a significant component of the immune state (see above). Moreover, a single dose of vaccine does not produce a protective level of antibody in all subjects, protective levels of antibody might not be sustained over the entire epidemic period, the antibody response in the very young might be inadequate owing to the lack of a prior priming infection, and the antibody response in the elderly could be impaired because of loss of immune responsiveness21. It is these problems that have encouraged research into other approaches to vaccine development. 403
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Because it is the immune response to the influenza virus HA that is the most important of the immune reactions, strategies for influenza vaccine formulation include: HA as a recombinant protein22; HA produced by a baculovirus expression system23; influenza virus grown in Vero cells, which yields a vaccine virus more akin to the virus circulating in man than virus grown in eggs24; and the use of a variety of HA epitopes to give chimeric proteins that could expand the antigenic repertoire of the vaccine and the immune response to it25. It is still early days for many of these approaches, but most represent alternative ways of producing HA that would be pharmacologically more desirable because it is a purer product and cheaper to produce, or expand the volume to overcome the limitations of inactivated vaccine production outlined above, but do not compensate for the limitations listed. Alternatively, the last two decades has seen the development of a large number of adjuvant or carrier systems that, combined with inactivated influenza virus vaccine, potentiate the immune response; a recent review of these systems lists more than 30 adjuvants, and numerous carriers and vehicles, each of which includes a number of variants26. Some adjuvants are potent enhancers of antibody; others can direct the immune response towards either an antibody or a CMI response27; and a third group can promote both reactions26. Most of the studies with these agents have been performed in mice, and some of the more promising systems have been translated to volunteer studies.The results of these latter studies have been disappointing, because some of the systems are too toxic for human use28 and others have not enhanced the antibody response to the same degree as seen in animal studies because species differ29. To date, the only licensed additive for inactivated influenza vaccine is aluminium salts, which are included more to limit toxicity than to provide enhanced immune response17,30,31; two other additives have recently received limited licence. The lack of conformity between animal and volunteer studies could be because of differences in the immune responses of different species, as mentioned above, but might also result from experimental design. Most animal experiments have been performed in nonprimed animals, whereas volunteer studies are in primed individuals; experiments in animals should therefore be conducted in primed animals to model the human situation. Much work remains and a large number of adjuvant systems are under current investigation; however, data are not available, and many research results in this competitive field are probably withheld for commercial reasons. Live attenuated vaccines The reservations that surround inactivated influenza virus vaccines have been an impetus for the development of live attenuated vaccines, which theoretically offer several advantages32,33. 404
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For example, live vaccines can be given as sprays or drops, induce local IgA and CMI responses and are suggested by some volunteer studies to induce a more solid immunity to challenge virus infections33,34. In addition, live virus vaccines might induce a more sustained and broader immune response, probably by inducing the more widely reactive IgA antibody and CMI (Refs 35–37). The logistics of producing a live influenza virus vaccine are more complicated than for inactivated vaccine, and, despite intensive research performed during the last 50 years, no live influenza virus vaccine has yet been licensed for use in man in Western countries. The history of this research is pitted with disappointment, but contains elements of promise. Early attempts to produce host-range variants as live, attenuated influenza vaccines ceased when these strains frequently failed to replicate sufficiently, induced disappointing immune responses and were unpredictable in the time it took for attenuation to occur32,33. Temperature-sensitive variants of influenza virus were developed as vaccines by two groups, but neither was successful33. Attenuated viruses that were a reassortant of a wildtype virus and a stable attenuated virus, and contained the surface antigens of the wild-type virus and the other properties of the attenuated virus, were found to be unacceptable, with one exception; most of these strains were unstable in seronegative volunteers32. The one exception is reassortants of wild-type virus and the attenuated strain, influenza virus A/AA/6/60. These reassortants are immunogenic and safe by all the criteria examined, and remained attenuated in all the volunteer studies performed in the past 30 years32. It is hoped that these vaccine viruses will form the first generation of live influenza viruses to be licensed in the Western world. However, this assumes that the long history of the safety of these virus reassortants will permit rapid development and licensing in time for an anticipated epidemic, because should any of these viruses be considered for a new vaccine and therefore require the complete spectrum of clinical studies prior to release, the time taken for development will be longer than the relevance of the vaccine strains to an anticipated epidemic. In addition, some authorities hold theoretical reservations about these viruses: with more than one strain of attenuated virus probably required for each complete vaccine, infection by one strain might interfere with another to limit the immune response33; reassortment might occur between a vaccine virus strain and a wild-type strain to produce a hybrid strain of unknown virulence; and attenuated strains need to be rigorously tested in infirm and aged people, who represent a target group for immunization, to ensure safety. In parallel with the developments listed above, other strategies to produce live influenza virus have been considered. First, by the use of reverse genetics and site-directed mutagenesis, it
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is possible to produce a virus strain that does not permit reversion to a virulent type. It is also possible to produce chimaeric viruses by the same strategy and this would preclude the problems of interference and could cover a broad range of virus variants38,39. However, these techniques are not simple, and, although they promise much in theory, they have yielded little in practice. Alternatively, influenza HA could be transfected into a virus vector, such as vaccinia or retroviruses, to form a live vaccine; or influenza vaccines could take the form of a DNA vaccine, a strategy that has been shown to be markedly effective for other infections40,41. The outcome of these initiatives lies in the future, but the hopes for the present are that live reassortant virus vaccines based on the attenuated strain A/AA/6/60 will satisfy the licensing authorities.
days after virus infection, and is at a maximum titre two to three weeks post-infection42; after this time, titres decline and antibody is rarely found more than two months after infection17. In addition, several researchers have reported that intranasal immunization induces protection against subsequent virus infection43–45, but this has not been confirmed in other studies46. Conversely, intranasal vaccines are relatively poor inducers of serum antibody responses, and it is the serum antibody response to the virus HA that constitutes the most important parameter of the immune state6,9. Despite these observations and contradictions, research in this area has been very active, particularly in the use of carrier systems in conjunction with inactivated virus vaccine, which might offset some of the disadvantages mentioned above.
Inactivated intranasal vaccines The above summary of influenza virus vaccines suggests: that improvement in the efficacy of inactivated vaccines given by injection rests on the use of an appropriate carrier or adjuvant system; that the future for live, attenuated influenza vaccines depends on safety considerations and licensing authorities; and that theoretical vaccines based on reverse genetics or DNA strategies are possible, but are for the future. The present vacuum has been partially filled by research into other strategies. One of these has been the development, during the last decade, of inactivated vaccines given intranasally.This approach has several attractions: inactivated vaccine given intranasally is probably more acceptable to vaccinees, and possibly less reactogenic; the vaccine would stimulate a local IgA antibody response, which has been shown to be an important parameter of immunity to subsequent infection; and, because of ease of administration, such a vaccine could possibly be used repeatedly should the local immune response be of limited duration. Nasal-wash IgA antibody appears in secretions at four to seven
Inactivated influenza vaccines in saline given intranasally to volunteers From the early days of influenza research, the contribution of local IgA antibody to immunity to influenza has been recognized47, and the limitations of inactivated vaccine given by injection to stimulate higher levels of immunity could be partly because of the failure of these vaccines to provoke a local response31. One strategy to correct this limitation is to give inactivated vaccine intranasally, as drops or as an aerosol, in the hope that this will stimulate both local and systemic antibody responses; six volunteer studies using this strategy are shown in Table 1. Following one dose of vaccine given intranasally, serum antibody responses were poor, and in some studies absent. The different results probably reflect the different sensitivities of the tests used to measure antibody, but the conclusion is that a single intranasal dose of inactivated vaccine does not induce a satisfactory serum antibody response. In contrast, a single dose of vaccine induces local antibody.This local antibody was equated, in one study, with protection against challenge
Table 1. Response of volunteers to inactivated influenza vaccine in saline given intranasally Vaccine
Number of dosesa Serum
A/ENG/72 (H3N2) A/HK/68 (H3N2) A/HK/68 (H3N2) Trivalent (H3N2; H1N1; B) Trivalent (H3N2; H1N1: B) Trivalent (H3N2; H1N1: B) Control (trivalent; S/C) aDoses bAt
1 1 2 2 2 3 1
± + ++ ++ ++ ++ ++
Antibody responseb Nasal wash ++ ++ ++ ++ ++ ++ ±
Protection
Refs
+ NT NT + + NT +
48 49 49 50 51 50 17
given one month apart.
one month postimmunization.
++, good response; +, response; ±, doubtful response. NT, not tested.
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virus infection given one month later, and, in another study, with protection against natural infection. The local antibody response was found to be maximal at two to four weeks postimmunization; after this time the titres declined, and antibody was not found three months after immunization. It was assumed that immunity had also disappeared by this time48. The above limitations might be overcome if intranasal vaccines were given in multiple doses; four of the studies reported in Table 1 show the results for volunteers given two or three doses of vaccine49–51. Following multiple doses given one month apart, nasal and serum antibody responses were greater than following one dose; in two studies protection was demonstrated after immunization. However, none of the studies reported the duration of protection or the local antibody response. Failure to demonstrate protection following intranasal immunization could be because of loss of the short-lived local antibody response17,46. Despite the limited number and extent of the studies that have been performed, the published results are encouraging; clearly, two doses of vaccine are superior to one, and, theoretically, further doses could be given to induce better and more sustained serum and local antibody responses. These questions require testing; however, it is with these goals in mind that researchers have looked to adjuvants to promote greater and more long-lasting immune reactions.
hibit the use of many of them for parental immunization in the human population28, but there remain a number that are the subject of active research. Some of these latter adjuvant or carrier systems have been selected and given intranasally to animals in conjunction with inactivated influenza vaccine; in addition, others might also be considered, because the problems of toxicity are less acute than when given by injection. Some of the adjuvants that have been investigated and published are listed in Table 2. These include: microparticulate resins for which size and charge are critical; immunity-stimulating complexes (ISCOMs), which, in animal studies, have been shown to be very powerful adjuvants for a range of vaccines given parenterally54; liposomes, which provide a carrier system for vaccines; protosomes prepared from Neisseria meningitidis; and the B subunit of toxins from Vibrio cholerae, Escherichia coli and Bordetella pertussis. In each case, it is of interest to record: how many doses of vaccine were given; the serum and local IgA nasal antibody response; the CMI response; and protection against challenge virus infection. All adjuvants provoked a local antibody response, although the results using liposomes were disappointing. Five of the six adjuvants induced a serum antibody response, indicating priming of a CD41 T-lymphocyte response not seen with a single dose of saline vaccine41; and four of the six studies reported short-term protection from challenge virus infection. Questions that remain include the duration of immunity. Because immunity to influenza in volunteers following vaccination is required to persist for at least a year, it is of interest to know if animals challenged two, four or six months after immunization remain protected. In addition, nasal wash antibody was demonstrated at two to four weeks after immunization,
Inactivated influenza vaccines in adjuvants given intranasally to animals Of the large number of adjuvant or carrier systems that have been identified as promoters of immune responses26, several have been tested in conjunction with inactivated influenza vaccine in animals, and some in man. Problems with toxicity pro-
Table 2. Immune responses to inactivated influenza vaccine with adjuvant given intranasally to mice Influenza vaccine virus
A(H1N1) 2 A(H1N1) A(H3N2) A(H3N2) A(H1N1) A(H1N1) A(H3N2) aMultiple bAt
Adjuvant
Microparticle resin Bordetella pertussis (B oligomer) ISCOMs Liposomes Vibrio cholerae (B subunit) Escherichia coli toxin B+ (B subunit; holotoxin) Neisseria meningitidis proteosomes
Cell-mediated responses
Protection (week p.i.)
Refs
Serum
Nasal wash
1 1 1 1 1,2 2
++ ++ ++ + NT ++
++ ++ ++ + ++ ++
– NT NT NT + NT
+ (NK) + (3) – (3) – (3) NT + (4)
52 53 54 54 55–57 58,59
3
++
NT
+
+ (4,8)
60
doses given at intervals of three to four months.
approximately four weeks postimmunization (p.i.).
++, good response; +, measurable response; –, no response. NT, not tested; NK, not given.
406
Antibody responseb
Number of dosesa
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and again the duration of local antibody response is of importance. Finally, it is noted that all the studies were performed in unprimed mice, and a direct translation to anticipated results in humans is not appropriate. Except for very young children, all volunteers will have experienced clinical or subclinical infection by influenza virus or received immunization; collectively, all volunteers are primed and results obtained in primed volunteers and unprimed animals might therefore be distinct. It is important to mimic the human situation and repeat the experiments reported in Table 2 in primed animals. Despite these reservations, the results are encouraging: some adjuvants can provide an enhanced serum antibody response, local antibody production, a CMI response and protection from challenge virus infection. Direct comparison of some of these systems with saline vaccines in which multiple doses can induce CD41 T-lymphocyte response (Table 1) would be of interest. Inactivated influenza vaccines in adjuvant given intranasally to volunteers Numerous adjuvants have been shown to amplify the immune response to inactivated influenza vaccines given intranasally to mice (Table 2), but few have progressed to volunteer studies. The results of two such studies are shown in Table 3; the same format is used as in Tables 1 and 2, thereby revealing the paucity of information that is available.The B subunit of toxins from a variety of bacteria have been shown to be potent adjuvants55–59. These act in a variety of ways, including relaxing tight junctions between epithelial cells, thereby allowing access of antigen to the underlying cells of the immune system, and promoting binding of antigen to cells. Inactivated vaccines in conjunction with the B subunit of E. coli toxin and containing minimal amounts of holotoxin have been studied in volunteers. In one study, increases in serum antibody titre were noted following two doses, given four weeks apart, and local nasal IgA was found; indeed, it was reported that the antibody
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responses were superior to those induced by saline vaccines alone61. However, results on the duration of the local antibody response and the CMI effect were not given, and immunity to challenge virus infection was not measured. In a second study, using trivalent inactivated vaccine in conjunction with the B subunit of E. coli toxin and delivered in virosomes, preliminary results reported an antibody response to all three influenza virus vaccine components; however, local antibody and CMI responses were not measured, no comparison was made with other vaccine strategies, and protection data were not obtained (Gluck, 1999, conference proceedings).These two recent studies represent two attempts to demonstrate efficacy for inactivated influenza vaccines with adjuvant given intranasally, but leave many questions unanswered. We await the results of further and more extensive studies with interest. Conclusion The recognized limitation of inactivated influenza virus vaccines given subcutaneously and the theoretical reservations for the safety of live attenuated influenza vaccines have given impetus to research to develop alternative immunization strategies. The importance of the local IgA antibody response has led researchers to consider immunization with inactivated vaccine in saline given by the intranasal route. This will probably require multiple doses and/or greater concentrations of antigen, but the strategy does stimulate a better local antibody response than inactivated vaccine given by injection. It remains to be seen if repeated doses will continue to recall local antibody, which is a short-lived response, or whether a local anergy develops. The experiments are important because the effects of immunization in man should last at least 12 months; however, studies in primed animals followed by volunteer studies are needed. The use of adjuvants in conjunction with inactivated vaccine should be both dose- and antigen-sparing, and studies in animal
Table 3. Immune response to inactivated influenza vaccine with adjuvant given intranasally to volunteers Influenza vaccine virus
Trivalent (H3N2; H1N1; B) Trivalent aIn
Adjuvant
Antibody responseb
Number of dosesa
Serum
Nasal wash
Cell-mediated responses
Protection (week p.i.)
Ref.
Escherichia coli B subunit + holotoxin
2 (4)
+
+
NT
NT
61
Escherichia coli B subunit + virosomes
2 (1)
+
NT
NT
NT
See text
parentheses: interval between doses, in weeks.
bComparisons
with other vaccines not given.
+, measurable response. NT, not tested. p.i., postimmunization.
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models support this view; however, animal studies should be designed to align with the primed status of volunteers, and extended to measure the duration of both the local antibody response and immunity. Finally, the development of a suitable adjuvant for use in conjunction with inactivated vaccine given intranasally could be of great value: one dose might be sufficient; the system could be antigen-sparing; a full spectrum of immune responses might result; and immunity might be longlasting. Again, further studies are awaited with interest. References 1
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Komase, K. et al. (1998) Vaccine 16, 248–254
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Powers, D.C. et al. (1995) J. Infect. Dis. 171, 595–599
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Levi, R. et al. (1995) Vaccine 13, 1353–1359
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Lakey, D.L. et al. (1996) J. Infect. Dis. 174, 838–841
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Hashigucci, K. et al. (1996) Vaccine 14, 113–119
Contributions to Pharmaceutical Science & Technology Today We welcome suggestions for short reports, opinion articles and full reviews for publication in Pharmaceutical Science & Technology Today. Potential authors should contact the Editorial Office in the first instance with a brief outline of the scope of the proposed contribution. Article proposals should be directed to the Editor at the address on page V.
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Intranasal immunization with inactivated influenza vaccine Chris W. Potter and Roy Jennings The development of improved vaccines against epidemic and pandemic influenza virus infection remains a priority in vaccine research. Killed vaccines given by injection are both cost-effective and induce immunity; however, their limitations are well known. Live vaccines have been in development for many years, but difficulties and safety concerns have prohibited their licensing in Western countries. However, the newer technologies of vaccine development, including DNA vaccines and attenuated virus vaccines produced by reverse
Many of the problems of vaccine development relate to the antigenic variability of the virus: a rapid mutation rate means that the immune responses to infection or immunization in one year do not necessarily protect against infection in a subsequent year4. Even with this major reservation, the available vaccines are not ideal, and this has led to a search for new strategies. It is with these strategies, and in particular intranasal immunization with inactivated vaccine, that the present review is concerned.
genetics, remain a hope for the future. With these problems in mind, emphasis has been given to the development of inactivated vaccines that are administered intranasally, either as repeated doses of saline vaccine or in conjunction with suitable carriers or adjuvants. This review describes these latter developments and concludes that this approach offers advantages and should be vigorously researched.
Chris W. Potter* and Roy Jennings Sheffield Institute for Vaccine Studies Division of Molecular and Genetic Medicine and Division of Child Health University of Sheffield Medical School Beech Hill Road Sheffield, UK S10 2RX *tel: 144 114 272 4072 fax: 144 114 273 9926 e-mail: [email protected]
402
▼ Data from the past 100 years record the oc-
currence of influenza in most countries in some years, and in some countries in most years.These outbreaks can be local or more widespread, and at intervals of 10–40 years a new virus subtype emerges from a focal point to spread quickly throughout the world to infect most people1. Influenza epidemics remain unpredictable in both time and severity, and the importance of the infection has been recognized for centuries. The virus was first isolated in the laboratory in 1933 (Ref. 2). Vaccines have been developed for more than 60 years3, and vast resources of money and manpower have been devoted to the study of this disease, including the efforts of researchers, physicians, diagnostic laboratories, epidemiologists, pharmaceutical companies and health authorities. Despite this, success in controlling influenza has been limited, and annual outbreaks resulting in numerous deaths continue to occur.
Immunity to influenza The antigens of the influenza virus particles that induce immunity can be identified through using conventional methods. Influenza viruses A and B contain eight RNA fragments, coding for several structural proteins with molecular weights of 10,000–90,000, that make up the virus particles; further proteins, designated nonstructural proteins, are synthesized in infectious cells, but are not present in the completed virions5. These proteins, and the immune responses directed by them, can be studied individually, and, although a response to many of them can be detected, it is the immune response to the surface glycoproteins – haemagglutinin (HA) and neuraminidase (NA) – that constitute the immune state. It follows that all influenza vaccines must contain a relevant HA, and preferably a relevant NA, in the form of whole virus, split virus, subunit virus, live virus or DNA, for immunity to result6. Cell-mediated immune (CMI) responses, including cytotoxic CD81 T lymphocytes that recognize the virus nucleoprotein expressed on the surface of cells containing replicating virus to effect cell lysis7, are transient, but may contribute to immunity, limit clinical infection and provide a broad, heterotypic immunity in the short-term7,8. Many studies have been directed towards identifying the basis of immunity to influenza.
1461-5347/99/$ – see front matter ©1999 Elsevier Science Ltd. All rights reserved. PII: S1461-5347(99)00194-7
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Unequivocal evidence indicates that a specific serum antibody to the virus HA, haemagglutination-inhibiting (HI) antibody, is the most important immune response, providing immunity to subsequent infection.Thus, susceptibility to challenge virus infection is inversely correlated to the serum HI antibody titre6,9, and passive transfer of HI antibody protects normal and immunodepressed mice from challenge virus infection10.The importance of serum antibody to NA, termed NI antibody, was demonstrated in 1968 when a new virus subtype caused a pandemic; this virus shared the same NA as the virus strains that had caused epidemics in the previous years, but possessed a completely novel HA. Thus, there were individuals at the time who possessed NI antibody but not HI antibody against the new virus, and infection was less common in these subjects than in those without NI antibody, and the higher the titre of serum NI antibody the less common was the infection11. Pure NA vaccine has been shown to induce immunity to clinical disease, but not to infection12. Studies on subjects with varying titres of local IgA antibody and similar titres of serum HI antibody indicate that those with nasal IgA are relatively immune to infection compared with those without this antibody, and the greater the titre of IgA antibody the more solid the immunity13. In addition, animals given anti-IgA lose immunity to challenge virus infection14. The place of CMI in protection remains contentious.There is clear evidence that a CD41 T-lymphocyte response is required for antibody production.This is supported by numerous experiments in naive animals that show that serum antibody does not develop in response to inoculation with a single dose of inactivated virus vaccine, which is a poor inducer of CD41 T cells. Cytotoxic CD81 T lymphocytes do develop in response to inactivated vaccine in primed animals or volunteers and following live virus infection, but this response is relatively short-lived15, although it can be recalled rapidly following later exposure to virus antigen16. The consensus is that CMI responses are required for antibody production and might be important in short-term immunity after viral infection; however, the most important contribution of CMI is to provide a mechanism for limiting the duration of disease. The evidence outlined above indicates that an influenza vaccine should contain the specific HA and NA to stimulate a serum HI antibody response, and this is the prime requirement. Immunity is further strengthened by the presence of serum NI antibody, local IgA antibody and, in the shorter term, by specific CD81 cytotoxic T cells. Inactivated vaccines Inactivated influenza vaccines have been used to induce immunity in volunteers for more than 60 years3. Many of the earlier vaccines were reactogenic, and this experience has left an unfortunate legacy; however, since 1960, when methods to purify
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virus particles became available, these vaccines have been well tolerated. The current inactivated vaccines are produced from a reassortant virus of the current infecting influenza strain and a strain that grows well in embryonated eggs, yielding a virus strain that contains HA and NA of the wild-type virus and which can grow to high titre. The virus is grown in embryonated eggs, harvested, purified by zonal centrifugation and inactivated with either formalin or b-propiolactone. The virus is then used either as a whole virus vaccine, a split virus vaccine in which the intact particles are broken down by chemical means, or a subunit vaccine in which the virus particles are disrupted and only the purified HA and NA form the vaccine17. In each case, the vaccine is standardized to contain approximately 15 mg of HA per dose and is given as a single inoculation, particularly to ‘at-risk’ subjects, which include the elderly, patients with chronic lung or heart disease and those with metabolic disorders18. Because influenza viruses mutate rapidly, the antigenic form in an epidemic virus must be anticipated months in advance to allow time for vaccine production and distribution; modern vaccines contain three virus strains, each at 15 mg of HA per dose, to cover expected infection. At-risk patients, despite earlier concerns that repeated annual injections are not advisable, should receive vaccine annually in anticipation of winter epidemics19. Numerous studies have shown that vaccine induces serum HI antibody to titres above 40 in the majority of vaccinees17; this titre is the reciprocal of the homologous virus antibody titre established by the HI test and is equivalent to a 50% level of protection against challenge virus infection6. Some vaccinees asked to report reactions to vaccine record local redness and soreness; however, these reactions are mild and do not persist beyond 24–48 h, and systemic reactions are rare17. Numerous studies, including both open and closed studies, in which immunized volunteers are given a challenge virus infection, indicate that inactivated influenza vaccines induce immunity in 60–90% of subjects20. One of the main reasons for these relatively disappointing results is the need for vaccine formulation at least five to six months in advance of the influenza season; continuous virus mutation, termed antigenic shift and drift, can result in an epidemic being caused by a virus variant that is distinct from the vaccine strain. In addition, inactivated vaccine given parenterally is a poor inducer of local immunity, which is a significant component of the immune state (see above). Moreover, a single dose of vaccine does not produce a protective level of antibody in all subjects, protective levels of antibody might not be sustained over the entire epidemic period, the antibody response in the very young might be inadequate owing to the lack of a prior priming infection, and the antibody response in the elderly could be impaired because of loss of immune responsiveness21. It is these problems that have encouraged research into other approaches to vaccine development. 403
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Because it is the immune response to the influenza virus HA that is the most important of the immune reactions, strategies for influenza vaccine formulation include: HA as a recombinant protein22; HA produced by a baculovirus expression system23; influenza virus grown in Vero cells, which yields a vaccine virus more akin to the virus circulating in man than virus grown in eggs24; and the use of a variety of HA epitopes to give chimeric proteins that could expand the antigenic repertoire of the vaccine and the immune response to it25. It is still early days for many of these approaches, but most represent alternative ways of producing HA that would be pharmacologically more desirable because it is a purer product and cheaper to produce, or expand the volume to overcome the limitations of inactivated vaccine production outlined above, but do not compensate for the limitations listed. Alternatively, the last two decades has seen the development of a large number of adjuvant or carrier systems that, combined with inactivated influenza virus vaccine, potentiate the immune response; a recent review of these systems lists more than 30 adjuvants, and numerous carriers and vehicles, each of which includes a number of variants26. Some adjuvants are potent enhancers of antibody; others can direct the immune response towards either an antibody or a CMI response27; and a third group can promote both reactions26. Most of the studies with these agents have been performed in mice, and some of the more promising systems have been translated to volunteer studies.The results of these latter studies have been disappointing, because some of the systems are too toxic for human use28 and others have not enhanced the antibody response to the same degree as seen in animal studies because species differ29. To date, the only licensed additive for inactivated influenza vaccine is aluminium salts, which are included more to limit toxicity than to provide enhanced immune response17,30,31; two other additives have recently received limited licence. The lack of conformity between animal and volunteer studies could be because of differences in the immune responses of different species, as mentioned above, but might also result from experimental design. Most animal experiments have been performed in nonprimed animals, whereas volunteer studies are in primed individuals; experiments in animals should therefore be conducted in primed animals to model the human situation. Much work remains and a large number of adjuvant systems are under current investigation; however, data are not available, and many research results in this competitive field are probably withheld for commercial reasons. Live attenuated vaccines The reservations that surround inactivated influenza virus vaccines have been an impetus for the development of live attenuated vaccines, which theoretically offer several advantages32,33. 404
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For example, live vaccines can be given as sprays or drops, induce local IgA and CMI responses and are suggested by some volunteer studies to induce a more solid immunity to challenge virus infections33,34. In addition, live virus vaccines might induce a more sustained and broader immune response, probably by inducing the more widely reactive IgA antibody and CMI (Refs 35–37). The logistics of producing a live influenza virus vaccine are more complicated than for inactivated vaccine, and, despite intensive research performed during the last 50 years, no live influenza virus vaccine has yet been licensed for use in man in Western countries. The history of this research is pitted with disappointment, but contains elements of promise. Early attempts to produce host-range variants as live, attenuated influenza vaccines ceased when these strains frequently failed to replicate sufficiently, induced disappointing immune responses and were unpredictable in the time it took for attenuation to occur32,33. Temperature-sensitive variants of influenza virus were developed as vaccines by two groups, but neither was successful33. Attenuated viruses that were a reassortant of a wildtype virus and a stable attenuated virus, and contained the surface antigens of the wild-type virus and the other properties of the attenuated virus, were found to be unacceptable, with one exception; most of these strains were unstable in seronegative volunteers32. The one exception is reassortants of wild-type virus and the attenuated strain, influenza virus A/AA/6/60. These reassortants are immunogenic and safe by all the criteria examined, and remained attenuated in all the volunteer studies performed in the past 30 years32. It is hoped that these vaccine viruses will form the first generation of live influenza viruses to be licensed in the Western world. However, this assumes that the long history of the safety of these virus reassortants will permit rapid development and licensing in time for an anticipated epidemic, because should any of these viruses be considered for a new vaccine and therefore require the complete spectrum of clinical studies prior to release, the time taken for development will be longer than the relevance of the vaccine strains to an anticipated epidemic. In addition, some authorities hold theoretical reservations about these viruses: with more than one strain of attenuated virus probably required for each complete vaccine, infection by one strain might interfere with another to limit the immune response33; reassortment might occur between a vaccine virus strain and a wild-type strain to produce a hybrid strain of unknown virulence; and attenuated strains need to be rigorously tested in infirm and aged people, who represent a target group for immunization, to ensure safety. In parallel with the developments listed above, other strategies to produce live influenza virus have been considered. First, by the use of reverse genetics and site-directed mutagenesis, it
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is possible to produce a virus strain that does not permit reversion to a virulent type. It is also possible to produce chimaeric viruses by the same strategy and this would preclude the problems of interference and could cover a broad range of virus variants38,39. However, these techniques are not simple, and, although they promise much in theory, they have yielded little in practice. Alternatively, influenza HA could be transfected into a virus vector, such as vaccinia or retroviruses, to form a live vaccine; or influenza vaccines could take the form of a DNA vaccine, a strategy that has been shown to be markedly effective for other infections40,41. The outcome of these initiatives lies in the future, but the hopes for the present are that live reassortant virus vaccines based on the attenuated strain A/AA/6/60 will satisfy the licensing authorities.
days after virus infection, and is at a maximum titre two to three weeks post-infection42; after this time, titres decline and antibody is rarely found more than two months after infection17. In addition, several researchers have reported that intranasal immunization induces protection against subsequent virus infection43–45, but this has not been confirmed in other studies46. Conversely, intranasal vaccines are relatively poor inducers of serum antibody responses, and it is the serum antibody response to the virus HA that constitutes the most important parameter of the immune state6,9. Despite these observations and contradictions, research in this area has been very active, particularly in the use of carrier systems in conjunction with inactivated virus vaccine, which might offset some of the disadvantages mentioned above.
Inactivated intranasal vaccines The above summary of influenza virus vaccines suggests: that improvement in the efficacy of inactivated vaccines given by injection rests on the use of an appropriate carrier or adjuvant system; that the future for live, attenuated influenza vaccines depends on safety considerations and licensing authorities; and that theoretical vaccines based on reverse genetics or DNA strategies are possible, but are for the future. The present vacuum has been partially filled by research into other strategies. One of these has been the development, during the last decade, of inactivated vaccines given intranasally.This approach has several attractions: inactivated vaccine given intranasally is probably more acceptable to vaccinees, and possibly less reactogenic; the vaccine would stimulate a local IgA antibody response, which has been shown to be an important parameter of immunity to subsequent infection; and, because of ease of administration, such a vaccine could possibly be used repeatedly should the local immune response be of limited duration. Nasal-wash IgA antibody appears in secretions at four to seven
Inactivated influenza vaccines in saline given intranasally to volunteers From the early days of influenza research, the contribution of local IgA antibody to immunity to influenza has been recognized47, and the limitations of inactivated vaccine given by injection to stimulate higher levels of immunity could be partly because of the failure of these vaccines to provoke a local response31. One strategy to correct this limitation is to give inactivated vaccine intranasally, as drops or as an aerosol, in the hope that this will stimulate both local and systemic antibody responses; six volunteer studies using this strategy are shown in Table 1. Following one dose of vaccine given intranasally, serum antibody responses were poor, and in some studies absent. The different results probably reflect the different sensitivities of the tests used to measure antibody, but the conclusion is that a single intranasal dose of inactivated vaccine does not induce a satisfactory serum antibody response. In contrast, a single dose of vaccine induces local antibody.This local antibody was equated, in one study, with protection against challenge
Table 1. Response of volunteers to inactivated influenza vaccine in saline given intranasally Vaccine
Number of dosesa Serum
A/ENG/72 (H3N2) A/HK/68 (H3N2) A/HK/68 (H3N2) Trivalent (H3N2; H1N1; B) Trivalent (H3N2; H1N1: B) Trivalent (H3N2; H1N1: B) Control (trivalent; S/C) aDoses bAt
1 1 2 2 2 3 1
± + ++ ++ ++ ++ ++
Antibody responseb Nasal wash ++ ++ ++ ++ ++ ++ ±
Protection
Refs
+ NT NT + + NT +
48 49 49 50 51 50 17
given one month apart.
one month postimmunization.
++, good response; +, response; ±, doubtful response. NT, not tested.
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virus infection given one month later, and, in another study, with protection against natural infection. The local antibody response was found to be maximal at two to four weeks postimmunization; after this time the titres declined, and antibody was not found three months after immunization. It was assumed that immunity had also disappeared by this time48. The above limitations might be overcome if intranasal vaccines were given in multiple doses; four of the studies reported in Table 1 show the results for volunteers given two or three doses of vaccine49–51. Following multiple doses given one month apart, nasal and serum antibody responses were greater than following one dose; in two studies protection was demonstrated after immunization. However, none of the studies reported the duration of protection or the local antibody response. Failure to demonstrate protection following intranasal immunization could be because of loss of the short-lived local antibody response17,46. Despite the limited number and extent of the studies that have been performed, the published results are encouraging; clearly, two doses of vaccine are superior to one, and, theoretically, further doses could be given to induce better and more sustained serum and local antibody responses. These questions require testing; however, it is with these goals in mind that researchers have looked to adjuvants to promote greater and more long-lasting immune reactions.
hibit the use of many of them for parental immunization in the human population28, but there remain a number that are the subject of active research. Some of these latter adjuvant or carrier systems have been selected and given intranasally to animals in conjunction with inactivated influenza vaccine; in addition, others might also be considered, because the problems of toxicity are less acute than when given by injection. Some of the adjuvants that have been investigated and published are listed in Table 2. These include: microparticulate resins for which size and charge are critical; immunity-stimulating complexes (ISCOMs), which, in animal studies, have been shown to be very powerful adjuvants for a range of vaccines given parenterally54; liposomes, which provide a carrier system for vaccines; protosomes prepared from Neisseria meningitidis; and the B subunit of toxins from Vibrio cholerae, Escherichia coli and Bordetella pertussis. In each case, it is of interest to record: how many doses of vaccine were given; the serum and local IgA nasal antibody response; the CMI response; and protection against challenge virus infection. All adjuvants provoked a local antibody response, although the results using liposomes were disappointing. Five of the six adjuvants induced a serum antibody response, indicating priming of a CD41 T-lymphocyte response not seen with a single dose of saline vaccine41; and four of the six studies reported short-term protection from challenge virus infection. Questions that remain include the duration of immunity. Because immunity to influenza in volunteers following vaccination is required to persist for at least a year, it is of interest to know if animals challenged two, four or six months after immunization remain protected. In addition, nasal wash antibody was demonstrated at two to four weeks after immunization,
Inactivated influenza vaccines in adjuvants given intranasally to animals Of the large number of adjuvant or carrier systems that have been identified as promoters of immune responses26, several have been tested in conjunction with inactivated influenza vaccine in animals, and some in man. Problems with toxicity pro-
Table 2. Immune responses to inactivated influenza vaccine with adjuvant given intranasally to mice Influenza vaccine virus
A(H1N1) 2 A(H1N1) A(H3N2) A(H3N2) A(H1N1) A(H1N1) A(H3N2) aMultiple bAt
Adjuvant
Microparticle resin Bordetella pertussis (B oligomer) ISCOMs Liposomes Vibrio cholerae (B subunit) Escherichia coli toxin B+ (B subunit; holotoxin) Neisseria meningitidis proteosomes
Cell-mediated responses
Protection (week p.i.)
Refs
Serum
Nasal wash
1 1 1 1 1,2 2
++ ++ ++ + NT ++
++ ++ ++ + ++ ++
– NT NT NT + NT
+ (NK) + (3) – (3) – (3) NT + (4)
52 53 54 54 55–57 58,59
3
++
NT
+
+ (4,8)
60
doses given at intervals of three to four months.
approximately four weeks postimmunization (p.i.).
++, good response; +, measurable response; –, no response. NT, not tested; NK, not given.
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Antibody responseb
Number of dosesa
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and again the duration of local antibody response is of importance. Finally, it is noted that all the studies were performed in unprimed mice, and a direct translation to anticipated results in humans is not appropriate. Except for very young children, all volunteers will have experienced clinical or subclinical infection by influenza virus or received immunization; collectively, all volunteers are primed and results obtained in primed volunteers and unprimed animals might therefore be distinct. It is important to mimic the human situation and repeat the experiments reported in Table 2 in primed animals. Despite these reservations, the results are encouraging: some adjuvants can provide an enhanced serum antibody response, local antibody production, a CMI response and protection from challenge virus infection. Direct comparison of some of these systems with saline vaccines in which multiple doses can induce CD41 T-lymphocyte response (Table 1) would be of interest. Inactivated influenza vaccines in adjuvant given intranasally to volunteers Numerous adjuvants have been shown to amplify the immune response to inactivated influenza vaccines given intranasally to mice (Table 2), but few have progressed to volunteer studies. The results of two such studies are shown in Table 3; the same format is used as in Tables 1 and 2, thereby revealing the paucity of information that is available.The B subunit of toxins from a variety of bacteria have been shown to be potent adjuvants55–59. These act in a variety of ways, including relaxing tight junctions between epithelial cells, thereby allowing access of antigen to the underlying cells of the immune system, and promoting binding of antigen to cells. Inactivated vaccines in conjunction with the B subunit of E. coli toxin and containing minimal amounts of holotoxin have been studied in volunteers. In one study, increases in serum antibody titre were noted following two doses, given four weeks apart, and local nasal IgA was found; indeed, it was reported that the antibody
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responses were superior to those induced by saline vaccines alone61. However, results on the duration of the local antibody response and the CMI effect were not given, and immunity to challenge virus infection was not measured. In a second study, using trivalent inactivated vaccine in conjunction with the B subunit of E. coli toxin and delivered in virosomes, preliminary results reported an antibody response to all three influenza virus vaccine components; however, local antibody and CMI responses were not measured, no comparison was made with other vaccine strategies, and protection data were not obtained (Gluck, 1999, conference proceedings).These two recent studies represent two attempts to demonstrate efficacy for inactivated influenza vaccines with adjuvant given intranasally, but leave many questions unanswered. We await the results of further and more extensive studies with interest. Conclusion The recognized limitation of inactivated influenza virus vaccines given subcutaneously and the theoretical reservations for the safety of live attenuated influenza vaccines have given impetus to research to develop alternative immunization strategies. The importance of the local IgA antibody response has led researchers to consider immunization with inactivated vaccine in saline given by the intranasal route. This will probably require multiple doses and/or greater concentrations of antigen, but the strategy does stimulate a better local antibody response than inactivated vaccine given by injection. It remains to be seen if repeated doses will continue to recall local antibody, which is a short-lived response, or whether a local anergy develops. The experiments are important because the effects of immunization in man should last at least 12 months; however, studies in primed animals followed by volunteer studies are needed. The use of adjuvants in conjunction with inactivated vaccine should be both dose- and antigen-sparing, and studies in animal
Table 3. Immune response to inactivated influenza vaccine with adjuvant given intranasally to volunteers Influenza vaccine virus
Trivalent (H3N2; H1N1; B) Trivalent aIn
Adjuvant
Antibody responseb
Number of dosesa
Serum
Nasal wash
Cell-mediated responses
Protection (week p.i.)
Ref.
Escherichia coli B subunit + holotoxin
2 (4)
+
+
NT
NT
61
Escherichia coli B subunit + virosomes
2 (1)
+
NT
NT
NT
See text
parentheses: interval between doses, in weeks.
bComparisons
with other vaccines not given.
+, measurable response. NT, not tested. p.i., postimmunization.
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models support this view; however, animal studies should be designed to align with the primed status of volunteers, and extended to measure the duration of both the local antibody response and immunity. Finally, the development of a suitable adjuvant for use in conjunction with inactivated vaccine given intranasally could be of great value: one dose might be sufficient; the system could be antigen-sparing; a full spectrum of immune responses might result; and immunity might be longlasting. Again, further studies are awaited with interest. References 1
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