Bench-to-bedside review: Vaccine protection strategies during pandemic flu outbreaks

Vaccination is the most effective means for the prevention of influenza, including pandemic strains. An ideal pandemic influenza vaccine should provide effective protection with the fewest number of doses in the shortest amount of time, and among the greatest proportion of the population. The current manufacturing processes required for embryonated chicken-egg-based influenza vaccines are limited in their ability to respond to pandemic situations - these limitations include problems with surge capacity, the need for egg-adapted strains, the possibility of contamination, and the presence of trace egg protein. Several vaccine strategies to circumvent the deficiencies intrinsic to an egg-based influenza vaccine are in various phases of development. These include the use of cell-culture-based growth systems, concomitant use of adjuvants, whole virus vaccines, recombinant protein vaccines, plasmid DNA vaccines, virus-like particle vaccines, and universal flu vaccines.


Current seasonal and pandemic infl uenza vaccines
Infl uenza vaccines have been in existence since the mid-1940s [4]. Since then there have been advancements in manufacture and purifi cation techniques, leading to modern vaccines with improved safety profi les and standardized potency. Broadly, there are two types of seasonal infl uenza vaccines currently licensed for use: parenteral trivalent inactivated vaccine (TIV), and mucosal (nasal) live attenuated infl uenza vaccine (LAIV). In the United States, nonadjuvanted TIV and LAIV are approved for use. In Europe, LAIV and both adjuvanted and nonadjuvanted TIV are approved for use. A separate LAIV vaccine is also licensed for use in Russia.
Twice each year, the World Health Organization uses data from the Global Infl uenza Surveillance Network to select three candidate viruses for the updated seasonal vaccine. Th e selected strains are the ones predicted to circulate during the subsequent season of each hemisphere's winter. Th e Northern Hemisphere strain selection is performed in February, the Southern Hemisphere selection in September. In recent years, the vaccine contains two infl uenza A viruses, H1N1 and H3N2 subtypes, and an infl uenza B virus. Once candidate strains are identifi ed, seed viruses are further adapted for highyield growth in chicken eggs through genetic reassortment techniques to produce the vaccine virus strain.
After optimization of growth conditions, manufacturers create bulk quantities of vaccine virus from inoculated embryonated chicken eggs. Th e vaccine is purifi ed by a standardized process of zonal centrifugation or column chromatography from virus-containing egg allantoic fl uid, and during this process the virus is inactivated by formaldehyde. Treatment with detergents disrupts the viral envelope and leads to split virion or subvirion vaccines. Prior to vaccine distribution, each batch or lot of vaccine is tested for sterility and potency, using highly standardized reference reagents to ensure the correct concentration of vaccine antigen. In reference to H1N1 pandemic vaccines, these biologic reagents may only be obtained from Australia's Th erapeutic Goods Administration, Japan's National Institute of Infectious Disease, the UK's National Institute for Biological Standards and Control, or the US Food and Drug Administration (FDA) [5].
A LAIV was fi rst licensed for human use in the United States in 2003. It is approved for use in healthy and immunocompetent individuals, aged 2 to 49 years. When administered intranasally, LAIV provides superior protection in children [6,7] compared with TIV, and results in herd immunity in children and adults [8]. Th e US LAIV backbone (or the master donor virus) was generated by serial passage of an infl uenza A strain (A/ Ann Arbor/6/60 H2N2) and an infl uenza B strain (B/Ann Arbor/1/66) at lower temperatures in primary chick kidney cells, resulting in viruses that are temperature sensitive, cold adapted, and attenuated [9,10]. Each of the three LAIV strains is prepared using reverse genetic reassortment. Plasmids containing six master donor virus genes and two wild-type virus genes, representing hemagglutinin (HA) and neuraminidase (NA), are electroporated into Vero cells to produce the vaccine seed strains [6]. On the other hand, a Russian LAIV has been in use since the mid-1970s wherein the master donor virus is based on a serial passage-derived, cold-adapted A/ Leningrad/134/57 H2N2 virus and the B/USSR/60/69 virus [11,12]. Th e Russian vaccine reassortant is produced by co-culturing the master donor virus with wild-type virus, and no reverse genetics is used.
Once generated, the bulk viruses for the vaccine are mass produced using embryonated chicken eggs. Th e viruses are fi ltered and concentrated, but not inactivated nor disrupted, such that the fi nal vaccine contains live attenuated viruses expressing the contemporary HA and NA of that season.

Current basis of infl uenza vaccine protection
Th e surface of the enveloped infl uenza virus is decorated by two main antigenic determinants, HA and NA, which play important roles in virulence and pandemic potential. As such, they are the primary antigenic target of infl uenza vaccines.
Strain-specifi c serum anti-HA antibodies prevent binding of the virus to host target receptors, and result in effi cient viral neutralization [13]. Vaccination that induces suffi ciently high amounts of anti-HA antibodies are necessary to protect an individual from infl uenza infection. A serum hemagglutination-inhibition assay is technically simple to perform, automatable, and represents the conventional means for assessing immunogenicity; a hemagglutination-inhibition titer ≥1:40 has traditionally defi ned seroprotection and has been associated with a >50% reduction in risk of infl uenza infection [14]. A viral neutralization assay, however, is a functional assay that is technically more diffi cult to perform and requires live viruses, and therefore may require a biosafety level 3 facility. Th e hemagglutinationinhibition assay typically correlates well with the viral neutralization assay.
Anti-NA antibodies may contribute to protective immu nity by blocking the viral NA from releasing replicating viruses and allowing the subsequent dissemination of the virus to other susceptible host cells. Although anti-NA antibodies can mitigate the severity of infl uenza infection [15][16][17][18], they alone do not neutralize the virus nor prevent infection [19].
Both inactivated parenteral and nasally delivered LAIV may induce anti-HA antibody, but LAIV may provide protection against infl uenza despite the absence of a serum anti-HA antibody response [6]. Since LAIV is mucosally delivered, secretory IgA may be elicited. Complexes formed by dimeric secretory IgA are potentially more effi cient in inhibiting viral entry than IgG or monomeric IgA [20]. High levels of anti-HA secre tory IgA antibodies can be detected in nasal washes within 2 weeks and may persist for 1 year [21]. No standard antibody assay, however, has been established for evaluating LAIV effi cacy. Licensure of the current LAIV was on the basis of signifi cant effi cacy in multiple studies and not immunogenicity.
Cell-mediated immunity probably plays an important role in the control and prevention of infl uenza infection, but the identifi cation of cell-mediated immunity correlates of protection has been elusive. Th e elicitation of humoral immunity requires a complex and carefully orchestrated interplay of the cellular immune system, and no single marker has suffi ciently predicted vaccine response.

Goals of pandemic infl uenza vaccines
A major antigenic shift resulting in a pandemic potential infl uenza virus is anticipated to cause a major threat to public health. Th is phenotypic change is predicted to result in higher morbidity and mortality -especially among segments of the population that are historically at lower risk for severe disease due to seasonal infl uenza, such as healthy young adults. In addition, pandemics have been typifi ed by higher transmissibility and succes sive waves [22]. Modern international travel, widespread migration, and fl uid borders all facilitate the more rapid spread of pandemic infl uenza viruses.
For these reasons, a pandemic vaccine should ideally possess certain characteristics. Th e vaccine should provide protection with the fewest number of doses (that is, a single dose) in the shortest amount of time, and among the greatest proportion of the population (for example, infants, elderly and immunocompromised people). In order to ensure that the population will accept vaccination, the vaccine must have a high degree of safety and little reactogenicity. Other considerations include vaccines that are temperature stable (do not require coldchain storage) and that avoid the need for needle and syringe delivery. In addition, rapid development and production of massive quantities of vaccine should maintain a consistent and reliable manufacturing process.
Th ese vaccines must be evaluated prior to approval for use under extraordinarily compressed timelines. Th e challenge for the US FDA and other national regulatory authorities is to ensure safe and eff ective vaccines in a timely fashion through the evaluation of clinical data to support licensure [23]. Th e US FDA can facilitate rapid approval of pandemic vaccines based on limited clinical studies on safety and immunogenicity if the manufacturer has a US-licensed seasonal infl uenza vaccine and is using the same manufacture process. Under this instance, the vaccine is considered a strain change. On the other hand, if the manufacture process has not gained previous US licensure, safety and eff ectiveness studies are required. Th e US FDA guidance for accelerated approval of pandemic vaccines, however, potentially permits the use of an 'acceptable surrogate marker of activity that is reasonably likely to predict clinical benefi t' [24]. Alternatively, the US FDA has the authority to grant Emergency Use Authorization of an unapproved product, provided some critical criteria are met, during a national public health emergency; this status ends when the emergency declaration is terminated. As an example, the agency recently issued Emergency Use Authorizations for the use of oseltamivir in patients <1 year old and for intravenous peramivir. Consequently, US pandemic vaccines are pragmatically constrained to licensed manufacture processes.
In the European Union, the European Medicines Agency has a slightly diff erent regulatory process, allowing a rolling review procedure for the submission of data as they become available, in comparison with the single formal application procedure of the US FDA. In Europe, some manufacturers have adopted the development of a core dossier or mock-up vaccine strategy. Th is approach includes the collection of preclinical, safety, and immunogenicity data on an index infl uenza virus that has not recently circulated among humans and thereby may mimic the novelty of a pandemic virus [25]. Using this strategy, novel 2009 H1N1 adjuvanted infl uenza vaccines and cell-culture-based vaccines were approved for use in the European Union.

Current infl uenza vaccines and inherent limitations
Th e current manufacture of most infl uenza vaccines is dependent on generating large virus stocks from eggs. Th e requisite supply of suitable eggs is subject to erratic production by stressed or ill chicken fl ocks, contamination, and other unpredictable events. Eggs need to be specifi c pathogen free, quarantined, and constantly monitored to make certain they remain disease free before entering the supply chain.
A vaccine virus should be optimally adapted to grow in eggs to ensure suffi cient virus yield. Typically, one egg leads to one dose of inactivated seasonal fl u vaccine. Th ere have been reports of growth yields as low as 20 to 50% with the novel H1N1 vaccines, compared with seasonal viruses (Center for Disease Control and Prevention, unpublished data). Wild-type avian H5N1 viruses were problematic because replication leads to killing of the chicken embryo. Th e global production capacity of infl uenza vaccine is estimated to be 300 million to 350 million doses annually or approximately 900 million doses of monovalent pandemic vaccine (assuming a single 15 μg dose) [26]. Only one-sixth of the world's population may therefore have the opportunity to be immunized.
Th e time required to prepare the vaccine from virus stocks to the point of fi lling and distribution of vials is a further limitation. Under normal circumstances, there is an 8-week to 12-week period following receipt of wildtype virus to the release of a safe reference vaccine virus to the manufacturers. Th e manufacturer may require a few weeks to generate high-growth reassortant viruses. Another 8-week to 12-week period may be required to produce the virus stocks, to concentrate and purify the antigen, and to fi ll vaccine. Lastly, each vaccine lot must be quality tested prior to release.
Reverse genetic techniques, using plasmid rescue, have enabled researchers and manufacturers to produce highyield viruses that express the relevant surface antigens, but remain nonpathogenic or attenuated. Th ese techniques have also been found to be suitable for largescale vaccine manufacturing [27].
Th e two major bacterial contaminants of concern are Salmonella and Campylobacter, both of which can colonize chickens and contaminate eggs. During the 1976 Swine Flu vaccine campaign there was an increased incidence of Guillain-Barré syndrome (GBS) [28], an ascending motor paralysis characterized by autoimmune demyelination. Although a link between an antecedent Campylobacter jejuni infection and GBS is known, this has not been established as the cause of GBS with the 1976 infl uenza vaccine [29]. Th e association of GBS and infl uenza vaccine has not been observed with subsequent infl uenza vaccines. A biologic mechanism for postimmunization GBS has been hypothesized to involve the synergistic eff ects of endotoxins (the product of Salmonella contamination) and vaccine-induced autoimmunity [30]. Th e presence of autoreactive antibodies against common cellular moieties of neurons (that is, gangliosides), however, has been reported to be associated with GBS [31]. An alternate etiology implicates sialylated HA complexes in the 1976 vaccine that may have provided the molecular mimicry leading to the development of anti-GM1 ganglioside antibodies, thereby leading to excess GBS cases [32]. Th e analysis of sialylation of HA in vaccines and the measurement of anti-GM1 antibody have therefore been proposed as a prelicensure requirement [33]. With the current H1N1 pandemic vaccines, there have not been reports of excess cases of GBS beyond the expected baseline rate.
A fi nal limitation of the current infl uenza vaccine is egg allergy. Th e manufacture process may cause trace amounts of egg protein to remain in the fi nal vaccine. For those people with serious egg allergy, vaccination is a contraindication. Th is further illustrates the need to have a pandemic fl u vaccine prepared via a diff erent substrate.

Vaccine technologies in development
Several vaccine strategies to address the critical needs of a pandemic vaccine are in various phases of development. Th ese include the use of cell-culture-based growth systems, concomitant use of adjuvants, whole virus vaccines, recombinant protein vaccines, plasmid DNA vaccines, use of virus-like particles, and universal fl u vaccines.
Cell-culture-based growth systems have been approved for use in some European countries. Th ese technologies use African Green monkey kidney (Vero), Madin-Darby canine kidney and other mammalian cell lines as the substrate for viral replication, rather than hen's eggs. Madin-Darby canine kidney cells have been routinely used for viral plaque assays and for clinical isolation of infl uenza viruses [34][35][36]. Th e virus yield using cell culture is comparable with that of eggs [34]. Cell culture off ers a reliable and fl exible production process, which can be performed using closed aseptic techniques. Th e process allows for growth of a broad range of authentic virus strains without the need for egg adaptation [37]. Several cell-culture-based infl uenza vaccines have been shown to be safe, well tolerated and immunogenic in children, healthy young adults, and even among the older population [37][38][39][40]. One limitation for rapid licensure of cell-culture-based vaccine is the perceived risk that mammalian cell lines have the potential for tumorigenicity and oncogenicity. Th e requirement for the presence of animal serum (or fetal bovine serum) in the cell culture medium also presents a special problem for US licensure. Animal serum must be ensured to be free of potential contamination with fungi, bacteria, viruses and agents of transmissible spongiform encephalopathies, and the serum must be readily available and undergo batch variation testing. Th e use of synthetic proteinbased media, rather than animal serum, may help minimize the risk of transmissible spongiform encephalopathies and viruses, but these techniques are complicated and currently the cost is prohibitive [34,41].
Adjuvants have the potential to boost the immunogenicity of infl uenza vaccines and thereby are a dosesparing strategy. Th e only adjuvant that is currently US FDA approved is based on mineral salts (for example, aluminum hydroxide or alum). Th e interest in more immuno stimulatory adjuvants gained momentum when an inactivated avian H5N1 vaccine was found to be poorly immunogenic [42] and the addition of alum provided little to no benefi t [43,44]. Oil-in-water emulsion adjuvant systems have been approved for use with inactivated infl uenza vaccines in Europe since 1997 (that is, MF-59). In 2009, however, the European Medicines Agency granted approval for ASO3 to be used with formulations of the H1N1 pandemic vaccine. According to the World Health Organization, among the 150 million doses of H1N1 pandemic vaccine distributed globally, 30% are adjuvanted formulations containing either MF-59 or ASO3; primarily in use in Europe and Canada [45]. Th ese adjuvants are safe, associated with mild and transient local reactogenicity, and are otherwise well tolerated [43,44,[46][47][48]. When combined with an inactivated avian H5N1 vaccine, MF-59 [46,47] and ASO3 [48] demonstrated superior immunogenicity compared with the unadjuvanted vaccine. Other immunostimulatory adjuvants that might prove safe and eff ective include saponins, immunostimulatory complexes, and innate immune receptor ligand/agonists (for example, monophosphoryl A, unmethylated CpG, mutant heatlabile enterotoxin, and fl agellin). Th ese adjuvants therefore hold the potential to stretch out existing limited vaccine supplies. Furthermore, adjuvants may induce more broadly protective immune responses; the elicited antibodies were cross-reactive against heterologous H5N1 strains [46,48]. Th ese heterotypic immune responses may be vital for protection against emerging clades and subclades of pandemic viruses [10].
Whole virus vaccines were originally abandoned because of the increased reactogenicity compared with subvirion vaccines [49]. Th e 1976 swine fl u vaccine was a whole virus vaccine, adding to the stigma of using whole virus vaccines. Inactivated whole virus vaccines, however, can elicit greater immunogenic responses than subvirion vaccines and generate cross-reactive antibodies against Chua and Chen Critical Care 2010, 14:218 http://ccforum.com/content/14/2/218 heterologous infl uenza strains [50][51][52]. Currently, a Hungarian-approved and a European Medicines Agencyapproved H1N1 whole virus vaccine are available.
Recombinant protein expression systems hold the promise of rapidly generating purifi ed subunit vaccines. One such vaccine is composed of recombinant HA from a Baculovirus expression system. Recombinant HA vaccines are highly purifi ed and contain no antibiotics or egg protein. Because of the higher concentration of antigens, they elicit stronger immune responses with less reactogenicity among healthy young and older adults [53][54][55]. A phase III trial of a recombinant HA vaccine was eff ective against culture-proven infl uenza -presenting 86% cumulative incidence reduction [55]. Nevertheless, the regulatory barrier that exists includes the concern for residual amounts of insect cell and Baculovirus proteins.
Recombinant infl uenza virus-like particles are another vaccine technology under clinical development. Th ese vaccines mimic the live virus but are unable to replicate, as they lack the internal machinery or genetic material necessary for replication. Th ese virus-like particles are assembled in insect or mammalian cells and simultaneously express HA and NA along with the matrix M1 protein [56]. Virus-like particles are strongly immunogenic and have been found to be protective in seasonal and highly pathogenic infl uenza virus murine challenge models [57].
Plasmid DNA-based vaccines are yet another promising vaccine technology still at early stages of development. Th is technology is based on plasmid DNA taken up by muscle cells (transfection) resulting in the expression of plasmid encoded protein [58]. Th rough direct interaction with B cells and antigen-presenting cells, the host immune system is stimulated as a result of this transfection [58]. An H5N1 plasmid DNA vaccine that encoded HA, nucleoprotein, and matrix protein M2 provided protection in mouse and ferret models of lethal infection [59]. Th e potential advantages of this technology include a shorter time for vaccine production, a nondependence on cell culture media, and theoretically eliciting both humoral and cell-mediated immune responses [58,59]. Th ese possible benefi ts have yet to be proven in human trials.
One of the ultimate goals of infl uenza vaccine research is to develop a universal vaccine that would provide durable and longlasting protection against all infl uenza A strains, rendering the need for annual vaccination obsolete [60]. One target is the ectodomain of matrix protein 2 (M2e), a highly conserved 23-amino-acid protein component of the virus envelope. Although M2e is a weak immunogen, after combining M2e with a carrier protein (such as hepatitis B virus core particles) the resulting anti-M2e antibody conferred protection in a mouse model of lethal infl uenza infection [61]. Other highly con served infl uenza virus epitopes are under consi dera tion as potential universal fl u vaccine candidates.

Pandemic vaccination of the population
As pandemic vaccines will probably be in short supply, it is paramount that a tiered system of apportionment is developed to identify people at increased risk of substantial morbidity and mortality. In conjunction with allocation, an aggressive campaign to implement the immunization of these at-risk groups will need to be prioritized. Who are the most susceptible? During seasonal epidemics, the predominance of severe disease aff ects the extremes of age -older people and young infants. With the current 2009 H1N1 pandemic, the majority of cases have occurred among the younger adult population (age <65), with only 5% of older people aff ected [62][63][64]. Nevertheless, the severity appears to be similar to seasonal epidemics, as one-quarter of the hospitalized patients had at least one underlying medical condition; these conditions include asthma, emphysema, diabetes, chronic cardiovascular disorders, chronic renal disease, neurologic disorders, and immunosuppression of varying etiology [63,65]. Pregnant women are at increased risk of complications [65]. As such, the young and pregnant women are among the highest priority for the current H1N1 immunization campaign. For US public policy, the guiding principles concerning vaccination are based on the recognition of groups at high risk of exposure, such as healthcare personnel, close contact with infants <6 months of age, and other related caregivers. Th e World Health Organization recognizes the variability in country-specifi c H1N1 epidemiology and access to vaccine and other infl uenza-related resources, but also recommends that healthcare workers be among the highest priority to protect the integrity of essential health infrastructure; country-specifi c conditions should dictate the prioritization of the other highrisk segments of the population to reduce transmission, morbidity, and mortality.
Prior to the initiation of massive immunization, another critical question must be answered. Th e optimal dose needs to be identifi ed, and this may depend on age and underlying medical conditions. Th e standard dose of the annual TIV contains 15 μg HA per virus strain. With the inactivated subvirion H5N1 vaccine, however, a 15 μg dose was insuffi cient [66] and two 90 μg doses separated by 28 days was necessary to achieve immunogenic responses among >50% of recipients [42,67]. Th erefore, when an infl uenza virus strain is completely novel -as in the H5N1 virus -multiple doses (that is, two or more doses) of vaccine may be necessary to achieve protection. Fortunately, the data show that a single 15 μg dose of the 2009 H1N1 vaccine is suffi cient to elicit seroprotection among >93% of the healthy young adults [68,69].
With regard to the current novel H1N1 pandemic, a handful of pandemic vaccines are being made available in record speed. In the United States, the rapid manufacture of the 2009 H1N1 monovalent vaccines required the open collaboration of the Department of Health and Human Services, academia, and industry at a level of intensity never before experienced. At the global level, the World Health Organization has relied heavily on close collaboration with industry partners and independent experts, such as the Strategic Advisory Group of Experts on Immunization, for the concerted response to the current infl uenza pandemic. At the time of writing, donated H1N1 vaccine is planned for distribution to 95 resource-poor countries. In the United States, approved vaccines are based on traditional manufacturing processes, although adjuvanted vaccines are under fi eld testing. In Europe and Canada, adjuvanted and cellculture-based vaccines are being used. Vaccination policies guiding these events have been informed by the existing limited data, and continuous epidemiologic surveillance is required to determine the effi cacy of the current vaccination campaign and to detect the presence of mutations.
In conclusion, pandemic infl uenza represents an unpre dictable and critical public health emergency. Vaccination remains the most eff ective means to prevent and control infl uenza infection. Th e current manufacturing process, based on chicken eggs, has inherent limitations. Next-generation infl uenza vaccines and other technologies are under development and provide the promise of improved protection.

Competing interests
JVC declares that he has no competing interests. WHC has been a paid consultant of AlphaVax, LigoCyte Pharmaceuticals, Integrated BioTherapeutics, and Toyama Chemical Co.