Influenza Virus: Immunity and Vaccination Strategies. Comparison of the Immune Response to Inactivated and Live, Attenuated Influenza Vaccines
Abstract
Influenza virus is a globally important respiratory pathogen which causes a high degree of morbidity and mortality annually. The virus is continuously undergoing antigenic change and thus bypasses the host's acquired immunity to influenza. Despite the improvement in antiviral therapy during the last decade, vaccination is still the most effective method of prophylaxis. Vaccination induces a good degree of protection (60–90% efficacy) and is well tolerated by the recipient. For those at risk of complications from influenza, annual vaccination is recommended due to the antigenic changes in circulating strains. However, there is still room for improvement in vaccine efficacy, long-lasting effect, ease of administration and compliance rates. The mucosal tissues of the respiratory tract are the main portal entry of influenza, and the mucosal immune system provides the first line of defence against infection. Secretory immunoglobulin A (SIgA) and IgM are the major neutralizing antibodies directed against mucosal pathogens. These antibodies work to prevent pathogen entry and can function intracellularly to inhibit replication of virus. This review describes influenza virus infection, epidemiology, clinical presentation and immune system response, particularly as it pertains to mucosal immunity and vaccine use. Specifically, this review provides an update of the current status on influenza vaccination and concentrates on the two main types of influenza vaccines currently in use, namely the cold-adapted vaccine (CAV) given intranasally/orally, and the inactivated vaccine (IV) delivered subcutanously or intramuscularly. The commercially available trivalent IV (TIV) elicits good serum antibody responses but induces poorly mucosal IgA antibody and cell-mediated immunity. In contrast, the CAV may elicit a long-lasting, broader immune (humoral and cellular) response, which more closely resembles natural immunity. The immune response induced by these two vaccines will be compared in this review.
Introduction
Influenza virus is a respiratory pathogen belonging to the family Orthomyxoviridae (from the Greek myxa, meaning ‘mucus’) [1]. There are currently described three types of influenza virus (A, B and C) which are distinguished by antigenic differences in two of their internal proteins, nucleoprotein (NP) and matrix protein (M). The three types of virus also differ in their pathogenicity and genome organization. Type A is found in a wide variety of warm-blooded animals (birds and mammals), whereas types B and C are predominantly human pathogens. Influenza A and B viruses are the two types that most commonly cause human disease. Influenza A viruses are subdivided further into subtypes based on the surface antigens, haemagglutinin (HA) and neuraminidase (NA). Today, 15 subtypes of HA (H1–H15) and nine subtypes of NA (N1–N9) have been found in influenza A viruses, and all subtypes are found in aquatic birds.
Influenza viruses are negative-stranded, segmented RNA viruses. Influenza A (Fig. 1) and B viruses contain eight viral RNA segments, whereas influenza C has seven viral RNA segments. The genomes of influenza A and B viruses code for at least 10 proteins and the C virus codes for eight proteins in vivo using different reading frames and alternative RNA splicing. Each RNA segment is encapsidated by the NP to form a ribonucleotide NP (RNP) complex. The RNPs are surrounded by a shell of M (M1), which is enveloped by a lipid bilayer derived from the host cell upon budding through the cell membrane. Radiating from the surface are the two surface glycoproteins: the rod-shaped HA and the mushroom-shaped NA. A third integral membrane protein is the ion channel formed by the M (M2) in influenza A viruses or NB in influenza B viruses.
A diagrammatic representation of influenza A virus showing protein and RNA composition. HA, haemagglutinin; NA, neuraminidase; M1, M2, matrix proteins; NP, nucleoprotein; NS, non-structural proteins; PA, PB1, PB2, proteins involved in virus replication.
Immunity to infection is mediated by antibodies to the viral surface antigens, HA and NA; these will be described in more detail. The crystal structure of both HA and NA have been determined, and there is a wealth of information available about their structure and function. HA is the main determinant of virulence of the virus and plays an important role in the viral life cycle. HA is responsible for attachment of the virus to the sialic acid-containing receptors on the host cell surface and also fusion between the viral and endosome membranes resulting in release of viral RNPs into the cytoplasm. The HA is a homotrimer of approximately 220 kDa and is a type 1 membrane protein that is anchored in the viral membrane by its C-terminus. The head of HA contains the receptor-binding site at the tip of each monomer and the five antigenic sites that bind neutralizing antibodies. The NA cleaves sialic acid and plays important roles in viral entry and release. The NA is a tetrameric glycoprotein of approximately 240 kDa and is a type 2 membrane protein that is anchored in the viral membrane by its N-terminus. The NA consists of a hydrophobic stalk and a globular head that contains the enzymatic and antigenic sites.
Influenza is continuously undergoing antigenic change to escape the host's acquired immunity by two mechanisms: antigenic drift and antigenic shift. Antigenic drift is the accumulation of mutations in all influenza gene segments, but the changes are particularly important in the surface glycoproteins (HA and NA), which are constantly subjected to selection pressure by the host's defence mechanism. Point mutations are caused by the inherent error rate of the RNA-dependent polymerase complex, which lacks proofreading ability. The influenza vaccine has to be reformulated almost every year to take account of the changing virus. Influenza A viruses undergo more rapid antigenic drift than influenza B viruses. Antigenic shift occurs at infrequent and unpredictable intervals, when the current influenza A virus disappears and is replaced by a new subtype with novel glycoproteins (always a novel HA and often a novel NA); the source of such viruses is thought to be lower animals (e.g. birds and pigs) [2]. Antigenic shift occurs as a result of genetic reassortment of the genome segments from different influenza A viruses in a doubly infected host cell (Fig. 2). Antibodies towards the previously circulating subtype do not cross-react with the new subtype.
There have been three pandemics during the last century, H1N1 in 1918 (Spanish flu), H2N2 in 1957 (Asian flu) and H3N2 in 1968 (Hong Kong flu), in addition the H1N1 virus reappeared in 1977 (Russian flu) and has continued to cocirculate with the H3N2 virus. Sequence studies of the 1918 virus show that this virus was genetically similar to a classical swine virus [3]. The antigenic shifts that occurred in 1957 and 1968 are thought to be due to pigs serving as a mixing vessel in southern China, the hypothetical influenza epicentre [4, 5]. The haemagglutinin (HA) of human influenza A viruses have a binding preference for cell receptors containing α2–6-linked sialic residues, whereas avian viruses bind to α2–3-linked sialic acid. The respiratory epithelial of pigs has receptors expressing both α2–3- and α2–6-linked sialic acid and is thus able to be infected by both human and avian viruses. This figure shows the suggested mechanism of antigenic shift resulting in the appearance of the influenza A H3N2 virus in 1968. This antigenic shift occurred through reassortment between human and avian (duck) influenza A viruses by coinfection of a mixing vessel (pig) where the HA (segment 4) and PB1 (segment 2) genes were replaced by genes of avian origin. Avian viruses can also be directly transmitted to man as has occurred recently (Table 1).
Epidemiology
Influenza is a globally important respiratory pathogen that causes nearly annual epidemics and occasional pandemics. Influenza epidemics generally occur in the winter months in the Northern hemisphere and in May–September in the Southern hemisphere. In the USA alone, influenza is responsible for substantial morbidity and mortality, with an average of over 100,000 hospitalizations and approximately 20,000 deaths annually [6–8]. In a pandemic, global spread of a new influenza A virus (containing a new surface HA unrelated to viruses circulating prior to the outbreak) results in increased morbidity and mortality. In the USA, the estimated cost of a future pandemic is between $71 and $168 billion, excluding the disruption to commerce and society [9–11]. Recently, a number of avian viruses have crossed the species barrier and directly infected humans (H5N1, H9N2 and H7N7), resulting in illness and, in some cases, death (Table 1) and presenting a possible pandemic threat. These viruses were not able to spread effectively from human to human.
| Year | Subtype | Country/region | Number of people experiencing illness | Number of deaths | Comments |
|---|---|---|---|---|---|
| 1997 | H5N1 | Hong Kong | 18 (serious illness) | 6 | Highly pathogenic H5N1 in poultry at wholesale and retail markets. All poultry destroyed in Hong Kong |
| 1999 | H9N2 | Hong Kong | 2 children (mild illness) | Antigenically and molecularly similar to virus circulating in quail | |
| China | 5 (mild illness; not laboratory confirmed) | ||||
| 2003 | H5N1 | Hong Kong | 2 (serious illness) | 1 | |
| H7N7 | The Netherlands (Belgium) | 83 (mild illness) | 1 | Highly pathogenic H7N7; affected birds slaughtered |
- The haemagglutinin (HA) is synthesized as a single polypeptide and cleaved into two subunits (HA1 and HA2) by the host cells proteases; this process is essential for viral infectivity. The HAs of the highly pathogenic H5N1 and H7N7 have multibasic cleavage sites allowing them to be cleaved by a number of enzymes [12].
Transmission and pathogenesis
Influenza is a respiratory virus that is spread from person to person, mainly by virus-laden droplets from coughing, sneezing or direct contact with an infected person. Influenza is characterized by the sudden onset of systemic (including fever, myalgia, headaches and severe malaise) and respiratory (nonproductive cough, sore throat and rhinitis) symptoms. The incubation period of influenza is usually 1–5 days, and the period of greatest communicability is during the initial 3 days of infection [13]; however, children can be infectious for longer periods. The virus can be shed before onset of symptoms and for as long as 7 days after onset of illness. Generally, uncomplicated influenza resolves itself within 1–2 weeks, although the cough may persist for more than 2 weeks. In high-risk subjects who have underlying medical conditions (e.g. cardiac, metabolic or pulmonary disease), influenza infection can lead to exacerbation of the underlying condition, secondary bacterial pneumonia, or, more rarely, primary viral pneumonia, which can result in hospitalization and death. During an influenza epidemic, one in 300 elderly people are hospitalized and one in 1500 elderly people die [14]. The rate of hospitalization in infants younger than 6 months of age is similar to that in high-risk adults, and an increased hospitalization rate is observed in children younger than 2 years of age [15].
Vaccine recommendations
Influenza causes disease in all age groups, but children have the highest infection rates, which may be as high as 30–40% in an epidemic [16], and more often have to be hospitalized [17]. However, serious illness, severe complications and death are most common in adults older than 65 years and those who have medical conditions that can be exacerbated by influenza infection. The group of people for whom influenza vaccination is recommended includes those who are most likely to be affected by complications of influenza (Table 2). Most Western countries have similar recommendations for influenza vaccination [19]. Vaccination is recommended for persons who are at increased risk from influenza complications, e.g. people older than age 65, those with chronic medical conditions such as cardiovascular, pulmonary, metabolic or renal disorders and immune-suppressed patients [11, 19]. Some countries also recommend vaccination of children on long-term aspirin therapy, and additionally, Belgium, Switzerland and the USA recommend vaccination of pregnant women in the second or third trimester (Table 2). Vaccination is generally recommended for residents of nursing homes and health care providers as well as household contacts of high-risk patients. In the USA, vaccination is also recommended for people aged 50–64 years and from 60 years of age in Austria, due to an increased rate of chronic medical conditions. Vaccination is also recommended in some countries to reduce the transmission of influenza for the following people: high-risk subjects, caregivers in nursing or chronic care homes, employees of assisted living facilities and home care workers of at-risk patients. No country recommends routine vaccination of children to reduce the rate of influenza-associated hospitalization, unless they have a high-risk condition, although in the USA, vaccination of children aged 6–23 months is encouraged.
| Target group | |
|---|---|
| Increased risk for influenza complications | People ≥65 years old |
| Residents of nursing or chronic care homes (not Finland, Greece, Portugal and Sweden) | |
| People with chronic cardiovascular or pulmonary disease including asthma | |
| People with chronic metabolic disorders including diabetes (not Iceland and Norway) | |
| People with renal dysfunction, haemoglobinopathy (not Iceland, Norway and Sweden) | |
| Children (6 months–8 years old) on long-term aspirin therapy due to an increased risk of Reye's syndrome | |
| (not Austria, Denmark, Germany, Greece, Iceland, Norway, Portugal, Spain, Sweden and UK) | |
| Immunosuppressed patients including infection with human immunodeficiency virus and medicated immunosuppression | |
| (not Austria, Finland, Iceland, Portugal and Sweden) | |
| Pregnant women in second or third trimester (only recommended in USA, Belgium and Switerzland) | |
| Possible underlying conditions | People 50–64 years old (only recommended in USA and from 60 years old in Austria) |
| People who can transmit influenza | Health care workers including doctors, nurses, hospital staff and emergency response workers |
| (not Denmark, Finland, The Netherlands, Portugal and Sweden) | |
| Household contacts of persons at high risk (only recommended in USA, Austria, Belgium, Greece, Ireland, Italy, Spain and Switerzland) |
- For further information, see [11, 18, 19].
The immune response to influenza infection
Both mucosal and systemic immunity contributes to resistance to influenza infection and disease [20–23]. Antibodies secreted locally in the upper respiratory tract are a major factor in resistance to natural infection [21–23]. Secretory immunoglobulin A (SIgA) is involved in protection of the upper respiratory tract and serum IgG in protection of the lower respiratory tract. The immune response induced by infection protects against reinfection with the same virus or an antigenically similar viral strain. Natural infection may lead to long-lasting immunity to the infecting virus, as demonstrated by the reappearance of the influenza A H1N1 subtype in 1977, when only subjects under the age of 20 years became infected [24]. Influenza virus undergoes frequent and unpredictable changes; therefore, after natural infection, the effective period of protection provided by the host's immunity may only be a few years against the new strains of virus circulating in the community [25].
The humoral immune system, including both the mucosal and systemic arms, plays a major role in immunity to influenza infection, and the cell-mediated immune response is particularly effective in clearing virus-infected cells. During infection of humans, antibodies are produced to all the major viral proteins [26, 27]. Antibodies to the surface glycoproteins, HA and NA, are associated with resistance to infection, whereas antibodies to the conserved internal antigens, M and NP, are not protective [28]. The cytotoxic T-cell (CTL) response is mainly directed against the M and NP proteins. Although this does not confer protection, it is important for the clearance of virus and recovery from illness.
Three terminologies are used for immunity to influenza A viruses. ‘Original antigenic sin’ is the recall of antibodies to the priming strain of influenza upon subsequent infection with another strain. ‘Homotypic immunity’ is protection provided to strains of virus within one subtype of influenza A. ‘Heterotypic immunity’ is cross-protection to another influenza A subtype conferred by previous infection with a different influenza A subtype; this type of immunity is normally weak in humans.
Humoral immunity
The humoral immune system produces antibodies against different influenza antigens, of which the HA-specific antibody is the most important for neutralization of the virus and thus prevention of illness. The NA-specific antibodies are less effective in preventing infection, but they lessen the release of virus from infected cells.
Mucosal antibody response
The mucosal tissues are the main portal entry of many pathogens, including influenza, and the mucosal immune system provides the first line of defence against infection apart from innate immunity (which will not be discussed here). SIgA and, to some extent, IgM are the major neutralizing antibodies directed against mucosal pathogens preventing pathogen entry and can function intracellularly to inhibit replication of virus [29]. Nasal secretions contain neutralizing antibodies particularly to influenza HA and NA, which are primarily of the IgA isotype [30] and are produced locally [31]. During primary infection, all three major Ig classes (IgG, IgA and IgM) specific to HA can be detected by enzyme-linked immunosorbent assay in nasal washings [20, 32], although IgA and IgM are more frequently detected than IgG. Both IgA and, to some extent, IgM are actively secreted locally, whereas IgG is derived as a serum transudate. In subjects who have a local IgA response, a serum IgA response also is observed. The local IgA response stimulated by natural infection lasts for at least 3–5 months, and influenza-specific, IgA-committed memory cells can be detected locally [32–34]. IgA also is the predominant Ig isotype in local secretions after secondary infection, and an IgA response is detected in the serum upon subsequent infection. The presence of locally produced neutralizing antibodies induced by live virus vaccine correlates with resistance to infection and illness after challenge with wild-type virus [20, 23]. However, resistance to influenza infection or illness is correlated with the level of local and/or serum antibody to HA and NA [28].
Serum antibody response
Serum anti-HA antibodies are the most commonly measured correlate of protection against influenza [35]. A protective serum antibody [haemagglutination inhibition (HI) titre ≥ 40] response can be detected in approximately 80% of subjects after natural influenza infection. B cells producing all three major Ig classes are present in the peripheral blood in normal subjects [36] and individuals undergoing influenza infection [37, 38]. In humans, serum antibodies play a role in both resistance to and recovery from influenza infection. The level of serum antibody to HA and NA in humans can be correlated with resistance to illness following experimental infection [20] and natural infection [39]. During primary infection, the three major Ig classes can be detected within 10–14 days. IgA and IgM levels peak after 2 weeks and then begin to decline, whereas the level of IgG peaks at 4–6 weeks. Whereas IgG and IgM are dominant in the primary response, IgG and IgA predominate in the secondary immune response [32, 40].
Cellular immune response
Cell-mediated immunity plays a role in recovery from influenza infection and may also prevent influenza-associated complications, but it does not seem to contribute significantly in preventing infection. Influenza-specific cellular lymphocytes have been detected in the blood and the lower respiratory tract secretions of infected subjects [41, 42]. Cytolysis of influenza-infected cells is mediated by CTLs in concert with influenza-specific antibodies and complement [43–46]. The primary cytotoxic response is detectable in blood after 6–14 days and disappears by day 21 in infected or vaccinated individuals [47]. Influenza-specific CTLs exhibit cross-reactive specificities in in vitro cultures; thus, they lyse cells infected with the same type of influenza but not with other types (e.g. influenza A but not influenza B virus) [42]. CTLs that recognize the internal nonglycosylated proteins, M, NP and PB2 have been isolated [42, 48]. There are great variations in the CTL reactivity pattern between subjects [46]. CTL memory cells (secondary response) exhibit a cross-reactivity pattern similar to primary response CTLs, which reach peak levels at day 14 and return to baseline after 6 months [47, 49]. The level of memory CTLs to influenza does not correlate with susceptibility to infection or illness following experimental influenza infection, but it does correlate with the rate of viral clearance from the respiratory tract [46]. There is a limited amount of human data on cellular responses available, but much information is available from the mouse model. Influenza infection induces a strong T-helper response, which plays an important role in stimulating antibody production against the virus [50]. The CTL response is cross-reactive between influenza A strains [51] and is important in minimizing viral spread in combination with antibody [52].
Treatment and prevention
There are two main methods of influenza prophylaxis: the use of antiviral drugs and vaccines. Several drugs are licensed in the USA for influenza prophylaxis: the M2 ion channel inhibitors (amantadine and rimantadine) and the NA inhibitors (zanamivir and oseltamivir) (Table 3). All of these except zanamivir also are licensed for treatment of influenza in the USA; however, zanamivir is licensed for prophylaxis in a number of countries including Australia, Switzerland, Gibraltar, Malta and Hong Kong. These antiviral drugs provide a useful adjunct to influenza vaccines, but vaccination remains the cornerstone of prophylaxis. At present, inactivated influenza vaccines are licensed worldwide; they first were licensed in the USA in 1945. In addition, cold-adapted (CA) influenza vaccines are currently licensed for use in Russia and have only recently been licensed in the USA. Therefore, this review will focus on inactivated and CA influenza vaccines (Table 4).
| Antiviral agent | Mode of action | Activity | Treatment | Prophylaxis (effectivity*) | Formulation | Side reactions | Other countries available for treatment |
|---|---|---|---|---|---|---|---|
| Amantadine | M2 inhibitor | Influenza A | +children ≥ 1 year | +(70–90%) children ≥ 1 year | Tablet/capsule/syrup | Generally mild CNS and gastrointestinal complications | Australia, Greece, Hong Kong, Ireland, The Netherlands, Norway, New Zealand, Switzerland, Singapore, South Africa, UK, Venezuela |
| Rimantadine | M2 inhibitor | Influenza A | +adults only | +(70–90%) children ≥ 1 year | Tablet/syrup | Generally mild CNS and gastrointestinal complications‡ | France (comprehensive information was not available) |
| Zanamivir | NA inhibitor | InfluenzaA and B | +children ≥ 7 years | −(84%) | Inhaled powder† | Generally mild diarrhea, nausea, sinusitis, nasal symptoms | Australia, Austria, Belgium,Canada, Denmark, France, Hungary, Ireland, Japan, Luxembourg, Norway, Portugal, Spain, Sweden, Switzerland |
| Oseltamivir | NA inhibitor | Influenza A and B | +children ≥ 1 year | +(82%) children ≥ 13 years | Tablet | Generally mild nausea, vomiting§ | Australia, Brazil, Canada, France, Hong Kong, Israel, New Zealand, Switzerland, UK |
- None of the antiviral agents are effective in preventing serious influenza complications. The information on countries where the drugs are available was supplied by the manufacturer of the individual antiviral drugs. For further information, see [11].
- * Effectivity in healthy adults.
- † Not recommended for patients with underlying airway complications.
- ‡ Rimantadine has a lower incidence of central nervous system (CNS) and gastrointestinal complications than amantadine.
- § Nausea and vomiting can be reduced by taking oseltamivir with food.
| Licensed vaccines | |
| Inactivated whole virus | Safe and immunogenic in humans |
| Inactivated split virus | Safe and immunogenic in humans |
| Inactivated subunit virus | Safe and immunogenic in humans |
| Vaccines undergoing licensure | |
| CA virus* | Safe and immunogenic in humans |
| Experimental vaccines | |
| Purified NA vaccine | Well tolerated and antibody to NA induced in humans, reduced viral replication and severity of illness [53] |
| Highly purified HA | Similar antibody response to subunit vaccine but lower [18] |
| Virosomal vaccine | Unilamellar virosome containing HA and NA, intranasal spray, immunogenic (serum HI, IgG and nasal-wash IgA). Well tolerated in adults and children [54–56] |
| Proteosome | Split influenza virus formulated with proteosome, intranasal or intramuscular immunization mice protected viral challenge [57] |
| DNA vaccine | Plasmid expressing HA, NA, M and NP. Humoral and cellular responses in mice and primates [58] |
| Naked RNA | Replicons containing NP or HA, induce immune responses in mice [59] |
| Immunomodulators for IV, e.g. | |
| MF59 | Adjuvant (Tween/Span emulsion) for IV results in increased antibody titres [60] |
| QS21, QS-7 | Saponin-derived fraction-combined IV augments immune response [61] |
| Enterotoxin (LT) from Escherichia coli | Inactivated vaccine plus LT, intranasally immunized mice protected viral challenge [62] |
| Dehydorepiandrosterone | Factor involved in immunosenescence, augmentation of immune response in old mice [63] |
- HA, haemagglutinin; HI, haemagglutination inhibition; Ig, immunoglobulin; IV, inactivated vaccine; M, matrix protein; NA, neuraminidase; NP, nucleoprotein.
- * Licensed 2003 in the USA.
At present, there are two subtypes of influenza A (H1N1 and H3N2) and influenza B circulating in the community. Current vaccines are thus trivalent to provide protection against the strains currently circulating. Recently, circulation of influenza A H1N2 has been detected; this virus is a reassortant of H1N1 and H3N2 viruses, and thus, current vaccines provide protection from this virus [11]. The World Health Organization (WHO) has an extensive surveillance network across the world that antigenically types and sequences the current isolates circulating in the community. Twice a year, the WHO meets to update the vaccine strains to match the strains circulating in the community in each hemisphere. The vaccine is standardized to contain the HAs of these recommended strains. Annual vaccination before the start of the influenza season (October–November) is the most effective means of reducing the impact of influenza and its associated complications. The current vaccine is relatively inexpensive and effective, but influenza remains responsible for the highest mortality in the USA by a vaccine-preventable disease [64]. Even in years during which one or more of the strains is the same as the preceding season, vaccination is recommended because of a decline in immunity. Vaccination is recommended for persons who are at increased risk of influenza complications due to underlying medical conditions, persons older than 50 years, residents of nursing homes and nursing home and health care workers, so that transmission rates of influenza can be reduced (Table 1). Young children (6–23 months old) should also be vaccinated, if practical, to reduce the rate of influenza-associated hospitalization.
Inactivated vaccines
Inactivated vaccines (IVs) are produced by propagation of the virus in embryonated hens' eggs. The allantoic fluid is harvested, and the virus is concentrated and highly purified, then inactivated with formaldehyde or β-propiolactone. Influenza vaccines may contain trace amounts of residual egg proteins and thus should not be administered to persons who have anaphylactic hypersensitivity to eggs. The vaccine is available in whole, split (chemically disrupted) and subunit (purified surface glycoproteins) formulations, which are administered intramuscularly or subcutaneously. Whole influenza vaccine is more immunogenic and also is associated with more frequent side reactions. In children younger than age 9, split or subunit preparations are preferred to reduce reactogenicity, and two half doses are recommended given at least 1 month apart [11]. The degree of protection after vaccination is dependent on the antigenic match between the vaccine strains and those circulating in the community, the age of the vaccine recipient and his or her previous history of influenza [65]. IVs are 60–100% effective in the prevention of morbidity and mortality [15], but they may have reduced effect in young (immune-naive) and elderly (decreased immune function) and in years of poor antigenic match. The antibody response in elderly individuals is often lower than that in young adults, but underlying conditions in the elderly are thought to be responsible for lower antibody responses rather than age, because healthy, older people have a response similar to young adults [65]. Influenza vaccine is 30–70% effective in preventing hospitalization in elderly people [66, 67].
Safety concerns
Millions of doses of inactivated influenza vaccine are administered each year, and the vaccine has excellent safety and tolerance profiles [15], with very low number of adverse reactions reported. Mild local reactions consisting of tenderness and redness at the injection site often are observed after inactivated influenza vaccination and occur in up to 25% of elderly recipients and <50% of healthy adults [65].
Immune response to trivalent inactivated vaccine
Vaccination with IV results in both local and systemic responses. As early as 2–6 days' postvaccination, increases in the serum antibody response to trivalent IV (TIV) can be detected [36, 68]. Within 2 weeks of vaccination, 90% of vaccinees have protective antibody titres [10, 36, 65]. Children as young as 6 months can develop protective antibody levels after vaccination [69–72]. The antibody response peaks 2–3 weeks postvaccination in primed subjects [36, 73, 74], and then wanes over time and is generally twofold lower by 6 months' postvaccination [18]. The serum antibody response is dominated by influenza-specific IgG antibodies (particularly IgG1), with lower concentrations of IgM and IgA antibodies detected [36, 73, 75, 76]. Antibody induced after vaccination is type specific [65] but can be highly cross-reactive, giving cross-protection towards earlier as well as newer viral strains [74]. The split-virus vaccine stimulates a strong immune response towards the surface virus glycoproteins (HA and NA); in some cases, antibody is stimulated towards the internal virus proteins (M and NP) [77]. A local immune response is induced after TIV in the tonsils (a respiratory secondary lymphatic organ) [74] and in the saliva/oral fluid [74, 78] (Fig. 3). Predominantly, IgG and IgA influenza-specific antibody-secreting cells (ASCs) were detected in peripheral blood [36, 74] and tonsillar tissue, which peaked in numbers 1 week after vaccination [74]. In the oral fluid, SIgA1 dominated the antibody response with lower titers of SIgA2 [74, 78]. Juvenile diabetics had an equally rapid protective immune response after TIV as healthy individuals in a pilot study [81], but further studies are needed to confirm this finding in other at-risk groups. In young, nonprimed children, the systemic immune response was dominated by IgM (which peaked later than IgG in adults), with a very low systemic IgA response and no or very little SIgA found in the saliva/oral fluid [76, 77]. The priming status is also important for the subclass of IgG and IgA produced [76].
A simplified overview of the immunological processes occurring in connection with parenteral influenza vaccination. After injection, the influenza vaccine components are transported to draining lymph nodes in the armpit, either as free antigen, opsonized or carried by dendritic cells [(or other antigen-presenting cells (APCs)]. Here, they induce an immune response. Elevated levels of influenza-specific serum antibody levels and peripheral blood lymphocytes are detected systemically after vaccination and most likely originating from the draining lymph nodes in the armpit [36, 73]. Influenza-specific plasma cells may also migrate to local lymph nodes as exemplified by the tonsils [73] but not to the local mucosal surfaces [79]. Parenteral influenza vaccination also mounts an antibody response in the saliva [73, 74], but the origin of these antibodies is not known but may come from tonsils and other local lymphoid tissue. CD-4-positive T-helper cells may also migrate from tonsils to lymph nodes in the armpit, participating in the present immune response [80].
Recently, we found that there naturally is a high basal level of influenza-specific ASC in the nasal mucosa [34], but this level is not influenced by vaccination with TIV [79]. In healthy subjects, treatment with the antiviral zanamivir did not alter the kinetics of the immune response to parenteral influenza vaccination [82], suggesting that the antiviral drug can be combined with vaccination in an effort to protect against influenza infection in periods of high influenza activity.
Mucosal vaccines
Influenza vaccines for delivery by the mucosal route have been available for many years but are not currently licensed in Western Europe and only recently been licensed in the USA. The benefit of such a method of delivery is that vaccination resembles more closely natural infection and avoids the need for injection.
The mucosal immune system
The surfaces of the respiratory, gastrointestinal and urogenital tracts, which are not covered by skin, are referred to as mucosa [83]. In total, the mucosa covers an area that is about 200 times larger than the skin. This large area is not as resistant to penetration as the skin, because it is often limited by a thin epithelial layer without keratinization. A number of pathogens initiate infection through the mucosal surfaces. The mucosal surfaces, particularly the respiratory and digestive tracts, are exposed daily to a wide variety of foreign organisms and antigens. Thus, they must be very effective in dealing with the large amounts of foreign antigens. The most important part of the immediate defence against pathogens in the mucosa is the innate immune system. The mucosa and associated lymphoid tissue are an attractive target for vaccine strategies, because they harbour the early stages of infection [84].
The mucosal surfaces provide several ‘layers’ of barrier, which potential pathogens must penetrate before initiating a successful infection. Firstly, there is secreted body fluids and mucus that contains hostile molecules and patrolling lymphocytes (e.g. macrophages). Intraepithelial lymphocytes (IELs) reside just below the epithelial surface, and further down beneath the basement membrane, a layer of mononuclear cells called ‘lamina propria’ is frequently found. These immune-competent cells constitute the first line of cellular immunity and are ready to combat potential pathogens that are approaching the mucosal surface or have penetrated the mucosal barrier. Foreign antigens may be taken up at the mucosal surfaces by specialized cells [e.g. M (microfold) cells] and actively transported to adjacent, organized lymphoid follicles. Lymphoid tissue localized adjacent to the mucosa is called the mucosa-associated lymphoid tissue (MALT) and may differ in size, organization and function, depending on its localization and which tissue it serves. MALT is therefore often distinguished by subgroups, e.g. nasal-associated lymphoid tissue, bronchial-associated lymphoid tissue and gut-associated lymphoid tissue.
The lamina propria and IELs consist mainly of resting memory T cells, but it also includes B cells, natural killer cells, macrophages and dendritic cells. A considerable portion of the T cells found in the mucosa express the γδ T-cell receptor and may be involved in conferring immune tolerance. Unlike the αβ subset of T cells, the γδ cells are not thymus dependent. The lymphocytes are guided to the mucosa by chemotactic components (chemokines) and interaction of selectins/integrins on the lymphocytes and addressins on local endothelial cells. The active homing factors (selectins and addressins) are lymphocyte and tissue restricted. Also, adhesion molecules on local tissue and lymphocytes are important in determining lymphocyte homing and distribution in the mucosa. Lymphocytes with certain antigen specificity often, but not exclusively, home to the site of first encounter with that particular antigen.
The majority of B cells in the mucosa are plasma cells secreting IgA. IgA is produced in large quantities every day (about 66 mg/kg body weight/day), and much of this is actively secreted into the body fluids. This part of the acquired immunity provides another barrier for pathogens, and it is therefore important in protection against a range of pathogens, including the influenza virus. In the absence of serum antibodies, it is thought that IgA may play a major role in determining resistance to influenza infection and illness.
Live, attenuated vaccine
The goal of live, attenuated vaccination is to induce a secretory and systemic immune response that more closely resembles the immune response observed after natural infection. Live, attenuated influenza vaccines induce broad mucosal and systemic responses. CA live, attenuated vaccines are administered intranasally, which results in limited viral replication in the upper and lower respiratory tract. The vaccine is easily administered intranasally (0.25 ml in each nostril) via a spray device that produces an aerosol of large particles, which deposits in the nasopharynx [85]. Previously, CA vaccines (CAVs) were administered by nasal drops; in young children, no difference was found in immunogenicity or reactogenicity between the two methods of delivery [86]. The dose of CAV required for infection of humans depends on the age, prior exposure to related strains and the level of pre-existing immunity in the recipient. Generally, a dose of CAV contains approximately 107 TCID50 (tissue culture infectious dose) of each of the three vaccine strains [87]. The currently available CAVs are attenuated, genetically stable, nontransmissible, safe, immunogenic and provide protective efficacy/immunity; thus, they fulfill the general requirements for use of CA reassortant vaccines in humans.
Production of live, attenuated vaccines takes advantage of the segmented nature of the influenza virus genome by reassorting a donor strain with a wild-type virus. Live, attenuated vaccines contain the genes encoding the surface glycoproteins (HA and NA) of the wild-type virus and the six remaining internal segments from the attenuated donor strain (PB1, PB2, PA, M, NP and NS) and are referred to as 6/2 reassortants. There are two master donor strains used in the USA to produce CAVs, one for production of influenza A strains, A/Ann Arbor/6/60 (H2N2), and another for production of influenza B strains, B/Ann Arbor/1/66. The master strains are CA, temperature sensitive and attenuated; thus, the virus has more limited replication at the warmer temperatures of the lower respiratory tract. The attenuation of live influenza vaccines is polygenic, reducing the possibility of reversion to virulence and involves all six genes donated by the donor virus for both influenza A and B strains [88].
CAVs are produced by infecting embryonated hens' eggs with the wild-type and donor viruses in the presence of antibodies to the surface glycoproteins of the donor strain to produce the required 6/2 reassortant. The recently developed helper-free reverse genetics system will, in future, allow the possibility of preparing CA influenza vaccine strains rapidly [89, 90]. Current CAVs are trivalent, and the viral strains in the vaccine must be updated yearly according to the WHO recommendations to provide protection against influenza strains circulating in the community. Interference between viral strains has been documented for bivalent and trivalent preparations with lower frequencies of viral shedding and/or serum antibody responses, especially for the influenza B vaccine [89, 91–93]. The mechanism of interference is unknown but can be overcome to some extent by a second dose of vaccine [94, 95].
Most recipients (over 75%) of CAV have a serum antibody response and/or shed virus after vaccination. There is a direct correlation between viral shedding and the induction of a specific antibody response after CA vaccination. In susceptible children and adults, CAVs are 78–100% effective at preventing influenza-associated illness after experimental challenge or natural infection occurring within 8 months of vaccination [87, 96, 97]. The effect of herd immunity was seen in unvaccinated staff and children in a Russian school that had high rates of vaccination of school children [98].
Safety concerns
There are a number of hypothetical safety concerns with CAVs that are not present with IVs [15]: (1) the site of administration is close to the central nervous system (CNS), which may increase the risk of vaccine-induced CNS complications; (2) the CAV could undergo spontaneous genetic change; (3) there is a possibility of contamination with other human virus when the reassortant between the CA master strain and clinical human sample occurs; (4) if simultaneous infection occurred with another influenza virus at the time of vaccination, it could result in a future pandemic strain, if the virus was an animal influenza virus. But with today's knowledge and new vaccine-preparation technology, potential risks have been significantly reduced.
Tens of thousands of doses of CA virus have been administered worldwide, and no severe side reactions have been reported in CA vaccinees [87, 94, 95, 99–101]. CAVs appear to be safe and nonreactogenic when administered at high doses to subjects who have previously experienced influenza infection [89].
Even in young infants and children who have no previous immunity, the reactogenicity of influenza A H1N1 and H3N2 strains is low, despite efficient viral replication in these individuals [88, 102, 103]. In naive subjects, the CA virus is shed for a longer duration and in higher titres; in a minority of young children, a short duration of mild respiratory, gastrointestinal and systemic symptoms is observed after the first dose of vaccine [103]. CA viruses are not easily transmissible, because the peak level of replication is lower than the human infectious dose and there are not many clinical symptoms of disease (e.g. coughing and sneezing) [88]. The donation of the six internal genes from the donor viruses appears to result in stable attenuation of the vaccine viruses [88].
Immune response to CAVs
A primary response to live CAV is characterized by serum IgA and IgM peaking 2 weeks postvaccination and declining by 4 weeks, whereas the IgG response peaks 4–12 weeks postvaccination and is sustained for at least 1 year. In the serum, IgG1 is the predominant IgG subclass induced by CA vaccination. CAVs induce nasal-wash antibodies, particularly IgA, which peak 2–11 weeks postvaccination and generally gradually declines by 6 months [23]; however, in vaccinated children, the nasal-wash antibody may persist for at least 1 year after vaccination [23, 103]. There is a positive relationship between serum (IgG and IgA) and nasal-wash IgG and IgA titres, with increased nasal-wash titres observed in subjects with higher serum antibody levels [105]. In addition, CAVs stimulate lymphoprofileration, interferon-γ secretion and CTL responses. Generally, children, particularly seronegative children, have more frequent serum antibody responses than adults after CA vaccination [95, 104, 106, 107]. Seropositive children also are more likely to develop a nasal-wash IgA response than haemagglutination inhibition antibody response after CA vaccination [105]. However, in infants younger than 6 months of age, significant increases in antibody were less frequently observed than in older infants [108]. The development of mucosal antibody may be a more important indicator of immunogenicity of CAVs than serum antibody [105].
Comparison of the immune response to CA and inactivated influenza vaccines
The two types of influenza vaccine in general stimulate different compartments of the immune system [15]. Vaccination with CA virus stimulates the local immune system, whereas immunization with IV stimulates the systemic arm. Resistance to influenza infection correlates with several immune parameters (as described above) but particularly with anti-HA antibodies in the serum and nasal wash [109]. The serum antibody response to influenza virus is commonly measured using the HI test; an HI titre of ≥40 is deemed protective [110], or alternatively, a fourfold increase in HI antibody titres is considered significant [111]. Whereas the correlate of protection has been clearly defined for serum HI titres, this is less clear for mucosal antibodies.
TIV produces a significantly higher and greater magnitude of serum HI, IgG and IgA antibody responses than CA virus [23, 112, 113]. The difference in induction of HI antibody titres by the two vaccines is most pronounced in recipients with no or low prevaccination antibody [15], and HI antibodies are induced twice as often in recipients of TIV than those receiving CA virus [15]. Although CA virus induced significantly lower levels of serum antibodies than TIV, it induces significantly higher levels of nasal-wash IgA [15, 85], e.g. approximately 50% of CA vaccinees have nasal-wash IgA compared with 25% of TIV recipients [15]. However, TIV is as effective as CA at stimulating nasal-wash antibodies, but IgG is the predominant class [23]. The levels of serum (IgG and IgA) and nasal-wash IgG remain elevated for at least 28 weeks after vaccination with CA or TIV vaccines [23]. In a monovalent challenge of subjects who had been vaccinated with CA virus or TIV, both vaccines reduced viral shedding, decreased the duration of viral shedding and reduced the severity of respiratory symptoms [22]. The two vaccines have a similar efficacy in preventing laboratory-confirmed influenza [15, 113]; the CAV was 85% effective and TIV was 71% effective. In a meta-analysis of 18 clinical trials comparing the use of TIV and CAVs encompassing 5000 vaccinees, it was found that although the two vaccines stimulate different arms of the immune system, they were similarly efficacious in preventing culture-positive influenza illness and had similar rates of side reactions [15].
The combination of CA and TIV is safe and well tolerated in the elderly [114, 115] and most frequently elicited serum IgG and nasal-wash IgA [109, 113]. By 3 months after vaccination, these levels had declined. In the elderly, the combined use of TIV and CAVs results in much greater protection from laboratory-confirmed influenza than the use of TIV alone [114]. The use of CAV in children (2–8 years old) significantly reduced the amount and the number of days of viral shedding after challenge, whereas a single dose of TIV did not reduce viral shedding [104]. The reduction in viral shedding and protection was associated with prechallenge nasal-wash IgA as well as the levels of serum antibodies (HI, IgG and IgA) [97, 104].
The response to IVs is strain- and subtype-specific, whereas CA may provide broader immunity against circulating strains [86, 89, 97, 113, 116, 117], including heterotypic immunity.
Target groups
In young children, CA may be more immunogenic than TIV [39, 89, 112, 116], because previous priming by natural infection is essential for production of a systemic response upon vaccination with TIV [75]. Children under the age of 9 require two doses of TIV to induce protective levels of serum antibody. In seronegative adults, CAVs have an infectivity rate of 75–85%[89] and result in greater protection from clinical illness and decreased incidence and titre of viral shedding than in recipients of TIV [89, 118]. Children probably play a vital role in the initiation and propagation of influenza epidemics, and immunization of children may help modify the course of an epidemic [119–122]. In working adults, the use of CAVs resulted in a reduction in laboratory-confirmed cases of illness [116], a significant reduction in severe febrile illness, days lost from work, health care visits and medication used [123]. In addition, in a recent study, 70% of healthy working adults successfully self-administered the CAV [123]. However, in the elderly, CA and TIV vaccines have reduced immunogenicity and efficacy [35, 114], especially CAVs that only induce short-lived local and systemic responses [109]. In this group, the combination of both TIV and CA virus increases vaccine efficacy [99, 113, 114, 117] and is required to produce a magnitude of response similar to young vaccinees [117]. The improved efficacy may be due to an enhanced cytotoxic lymphocyte activity upon coadministration of TIV and CAVs [99, 101]. To date, it is unclear whether CAVs are safe in immunocompromized patients.
Conclusion
Live CAVs are a promising approach to influenza immunization that may prevent initial infection with the virus. CAVs are immunogenic in children (particularly younger children) and young adults. However, in the elderly, the immune response to CA is modest, but in combination with TIV, it provides increased protection from influenza.
Acknowledgments
We thank Roland Jonsson (University of Bergen) for valuable discussion and Lori Lush and Carla Pellecchia (PharmD) for their assistance in the preparation of this review.
