Confronting Potential Influenza A (H5N1) Pandemic with Better Vaccines

Better understanding of host-virus interaction is essential to produce effective vaccines against influenza (H5N1) viruses.

Infl uenza A (H5N1) viruses are strong candidates for causing the next infl uenza pandemic if they acquire the ability for effi cient human-to-human transmission. A major public health goal is to make effi cacious vaccines against these viruses by using novel approaches, including cell-culture system, reverse genetics, and adjuvant development. Important consideration for the strategy includes preparation of vaccines from a currently circulating strain to induce broad-spectrum immunity toward newly emerged human H5 strains. This strategy would be a good solution early in a pandemic until an antigenically matched and approved vaccine is produced. The concept of therapeutic vaccines (e.g., antidisease vaccine) directed at diminishing the cytokine storm frequently seen in subtype H5N1-infected persons is underscored. Better understanding of host-virus interaction is essential to identify tools to produce effective vaccines against infl uenza (H5N1).

B ird fl u caused by the infl uenza A virus subtype H5N1
has spread with alarming speed across Europe, Africa, and parts of Asia in which the infection was not reported earlier. Establishment of the highly pathogenic avian infl uenza (H5N1) as an endemic virus within duck and poultry populations and its capacity to cross species barriers increase the possibility of adaptation to humans and a pandemic. Human infl uenza infections with subtype H5N1 viruses are often fatal. As of June 4, 2007, 309 laboratoryconfi rmed cases of human infection have been reported to the World Health Organization (WHO); 61% were fatal, mainly in persons 10-39 years of age (www.who.int/csr/ caculatordisease/avian_infl uenza/en). If a pandemic is triggered by transmissibility of infl uenza (H5N1) from person to person, millions of people could die, and economies would likely be crippled for 6-24 months.
In the event of a pandemic, vaccination against infl uenza (H5N1) could limit the impact of infection at a public health level. However, no evidence exists that available vaccines would be protective against the pandemic strain of the virus. We comment on some of the limitations of currently available vaccines and propose novel strategies to improve vaccine formulations against infl uenza (H5N1).

Host's Immune Responses to Infl uenza (H5N1)
The host response to infl uenza (H5N1) infection has not been defi ned, which has proven a considerable challenge in epidemiology and public health research. To develop effi cient vaccines, understanding how the virus interacts with the host in natural infection is necessary. Having insights into the hosts' responses to infl uenza (H5N1) would help defi ne targets for therapeutic intervention. Whether humans can develop immunity during a primary infection that would control replication and spread of subtype H5N1 viruses has been questioned (1). However, marked infl ammatory responses develop after infection with infl uenza (H5N1) in humans and other animals (2)(3)(4). This condition is associated with statistically signifi cant synthesis of various proinfl ammatory cytokines, such as tumor necrosis factor-α, interleukin (IL)-6, interferon (IFN)-γ, IL-1α, and chemokines, including IP-10, MIG, monocyte chemoattractant protein-1 (MCP-1, IL-8, and RANTES. i.e., regulated on activation, normal T-cell expressed and secreted). If this is the case, these observations are consistent with the possible induction of innate immune responsiveness in the persons infected with infl uenza (H5N1  (5). The death rate of ≈25% was half that of previously known outbreaks, and 5 mild or completely asymptomatic cases have been reported. One theory holds that milder cases have been occurring elsewhere but are not being recorded. Recently, 3 persons among 120 apparently healthy volunteers from the People's Republic of China, showed detectable virus-neutralizing antibody response to subtype H5N1 before vaccination (6). Moreover, pigs infected with subtype H5N1 have become asymptomatic in Indonesia. Are these signs of development of some degree of immunity to virus, containing its replication and thus causing milder infection in naturally infected mammals? Recently, clusters of bird fl u cases were reported in Western Java, Indonesia (7); fatal disease developed in 6 persons there from the same family. Two other family members became ill but survived. All the family members likely had similar levels of exposure because they all lived in the same household. Other cases of nonfatal infections have been seen in Thailand and Vietnam. Unfortunately, there is little information about the immune response to the virus in those who survived, which would be valuable for understanding the mechanisms of protection. Indeed, following up the persons (cohort) living in the same affected villages, presumably mostly not exposed to virus, should clarify whether the maintained response refl ects boosting through natural exposure. Persons with prior exposure, as measured by antibody or viral RNA at recruitment, would likely have substantially higher responses to the vaccine than those naïve at recruitment if the vaccinating antigen contains homologous or cross-reacting determinants. Conceivably, boosting the "natural" immunity is a desirable outcome to improve protective effi cacy of any vaccine approach. Additional studies are required to evaluate the merits of priming populations in advance of an infl uenza (H5N1) pandemic.
After initial hesitation about using a wide-scale program of poultry vaccination, some European and Asian countries have begun vaccination. Inactivated vaccines are widely used in poultry but lack of critical potency testing, standardization, and quality control has led to variable and suboptimal immune responses. Moreover, a legitimate concern remains that the fowl vaccinated by attenuated live viruses may survive the disease but still carry the virus; thus, they would continue to spread infl uenza (H5N1) silently at the fl ock level (8) or to humans who come into contact with them. Vaccination that resulted in low levels of seroconversion facilitated the emergence of the Fujian-like sublineage of infl uenza (H5N1) in poultry (9).
The immune responses elicited by subpotent vaccines may exert selection pressure that favors antigenic drift and shift ( Figure). Antigenic drift relies on the accumulation of mutations within the antibody-binding sites in the hemagglutinin (HA), neuraminidase (NA), or both that abrogate the binding of antibodies. This makes infl uenza A virus strains able to evade neutralizing antibody from prior infection or vaccination. Antigenic shift, which is seen only with infl uenza A viruses, is a more drastic change. It results from genetic shift by reassortment exchange of the HA, and sometimes the NA, with novel subtypes that have not been present in human viruses for a long time. Antigenic shift leads to replacement of circulating strains with new variants that are able to reinfect hosts immune to earlier types; the result is usually a pandemic. Antigenic shifts caused 2 of the major infl uenza A pandemics in the last century, including the 1957 subtype H2N2 and 1968 subtype H3N2 outbreaks (10).

Live Vaccines for Use in Humans
Most infl uenza vaccines used in the United States and Europe are produced in embryonated hens' eggs and are formaldehyde-inactivated preparations (11). Because highly pathogenic infl uenza (H5N1) subtypes may kill embryonated eggs, use of viruses that are no longer pathogenic, such as H5 (which lacks the polybasic cleavage site), to reduce the virulence of infl uenza (H5N1) vaccine strains so that these can be effi ciently propagated in eggs for vaccine production is feasible (10). Virus particles that lack the gene for the nuclear export protein or are defective for the matrix (M2) gene were used as live vaccines in animal models (12,13); however, whether these replication-defective vaccines will work in humans is not known. Live attenuated (cold-adapted) infl uenza vaccines have long been used in Russia, and a similar product has been approved for use in the United States (14). These vaccines will replicate in the host, and thus lower doses may be effective; however, the preexisting antibody to the virus is more likely to diminish the value of a live vaccine. Moreover, such live vaccines are reported to cause asthma-like reactivity in children (15). Monitoring live infl uenza vaccines is important because the risk for reversion to pathogenicity remains.
With the use of a technique known as reverse genetics, a prototype of infl uenza virus (H5N1) has been produced for the development of an inactivated subvirion vaccine. The gene segments encoding HA and NA were derived from A/Vietnam/2004, and all other genes were derived from the backbone (A/PR/8/34) virus, commonly used as a platform for infl uenza vaccine production. The HA gene was further modifi ed to replace the stretch of 6 basic amino acids at the cleavage site, and the resulting virus was avirulent in chickens. In a recent trial, healthy adult volunteers were given 2 intramuscular doses of this inactivated infl uenza (H5N1) vaccine. This split vaccine induced an antibody response predictive of protection in 54% of healthy adults tested, but only when given intramuscularly at high doses (two 90-μg shots) (16). The large amounts needed (2 doses of vaccine, each 6 times the dosage of that used in a standard infl uenza shot) means that hundreds of millions of doses are needed to tackle a pandemic. Dose-sparing approaches, including the use of an effi cient nontoxic adjuvant to boost persons' immune responses, may improve the vaccine. Another trial was performed with 300 healthy participants 18-40 years of age, in which aluminum hydroxide adjuvant was used with similar split-virus vaccine (17). However, the alumadjuvanted vaccines did not improve the immunogenicity or percentage of seroconversion at lower vaccine doses and only slightly improved immunogenicity at the 30-μg dose. This diffi culty underscores the importance of vigorous fundamental research to address the question of how to increase the immunogenicity of such vaccines, whether by better antigen presentation or by choosing alternative routes of administration, so that lesser amount of antigen could be given to induce protective response. The present annual global production capacity is ≈300 million doses of trivalent vaccine containing 15 μg HA per strain. This is equivalent to 900 million doses of monovalent vaccine, a quantity markedly insuffi cient for the world's 6.5 billion people. Clearly, dose-sparing formulations are urgently needed.

Inactivated Vaccines for Immunizing Humans
To test the hypothesis that whole-virion would be more immunogenic than conventional split-virion or subunit vaccines and may be adaptable to the antigen-sparing strategy, an inactivated, monovalent infl uenza A (H5N1), whole-virion vaccine was prepared from a highly virulent strain A/Vietnam/1194/2004 strain by removing the polybasic amino acids at the cleavage site, making the virus no longer pathogenic. The seed virus was grown to a high titer in embryonated eggs, inactivated with formalin, and purifi ed. These viruses were then adjuvanted with aluminum hydroxide and used in a phase 1 trial (6). The highest immune response of 78% seropositivity was observed in the group given 2× 10 μg HA, which is equivalent to that elicited by higher doses of nonadjuvanted (90 μg) or adjuvanted (30 μg) split-virion vaccines (16,17).
Not knowing which particular genetic variant will sustain human-to-human transmission makes our ability to formulate a vaccine in advance all the more diffi cult. An inactivated vaccine that induces not only high levels of neutralizing antibody to surface proteins but also CD8 Tcell response against well-conserved antigens derived from internal viral proteins might provide superior protection in an epidemic or pandemic. In cases of established intracellular infl uenza A infection, infected cells are mainly eliminated by effector CD8+ T cells (CTLs) (18). Any vaccine that will induce and direct these CTLs to the site of infection and generate a long-lasting memory response will be more effective for mounting protection against a pandemic form of infl uenza (H5N1). Inactivated vaccines need to be presented to the host's immune system with an appropriate adjuvant, but inactivated vaccines that use an adjuvant currently approved for human use (alum or MF-59) usually have lower immunogenicity than live attenuated vaccines (10). Therefore, the pursuit for other nontoxic adjuvants, including TLR ligands and agonists that could effectively activate dendritic cells for the presentation of viral antigens to CD4 and CD8 T cells, should vigorously be continued. Use of cytokines such as IL-12 or IL-18 may enhance the immunogenicity of antiviral vaccines. Recombinant fowlpox vaccines coexpressing HA of subtype H5N1 and chicken IL-18 have been shown to induce complete protection in vaccinated chickens (19). Use of adjuvants may enhance broader cross-reactive immune responses among infl uenza viruses (20).

Vaccines that Generate Broad-spectrum Immunity
The evolution of many sublineages of infl uenza (H5N1) with antigenic diversity in Southeast Asia and southern China favors the wisdom of developing broadly cross-reactive vaccines for protection against an epidemic or pandemic (21). Genetically engineered viruses could be constructed; these would express several variant antigens or determinants, thereby generating a broader immune response. The goal would be to develop vaccines that would induce broad-spectrum immunity-conferring protection to infl uenza including subtype H5N1. Ferrets vaccinated with A/PR/8/34 single-gene reassortants that differed only in their H5s were protected against a lethal challenge with A/Vietnam/1203/04 virus, suggesting generation of crossprotection (22). Vaccination of mice with a live attenuated infl uenza vaccine or an alum-adjuvanted inactivated infl u-enza vaccine based on a related H5 HA from a nonpathogenic avian infl uenza virus, A/Duck/Pottsdam/1042-6/86 (H5N2), limited the disease severity and reduced deaths following challenge with a current highly pathogenic infl uenza (H5N1) (23). Such cross-protective vaccines may provide clinical protection and prevent deaths in the early stages of a pandemic.
Genes of highly conserved proteins such as the nucleoprotein or M2 proteins could be included in adenovirus vector-based vaccines because immune responses against these infl uenza viral antigens provide protection in animal models (24,25). Recently, human adenoviral vector-based HA subtype 5 infl uenza vaccine induced protection in mice against infl uenza (H5N1) viruses isolated from humans (26,27). However, pre-existing immune response to human adenoviruses could be a potential problem in the generation of immune response against a foreign gene of interest. Delivering the vaccine nasally could largely overcome this problem because there appears to be no pre-existing immunity in the upper airways. Moreover, a robust CD8 T-cell response would likely be fl exible and able to fi ght infl uenza (H5N1).
Ideally, we need an effective vaccine for persons of all ages. However, if the vaccine is in short supply, priming fi rst those persons at high risk (e.g., young children, persons >50 years of age, healthcare workers) may be justifiable. During the early stages of an emerging H5 pandemic, such persons at high risk might be given an adjuvanted vaccine produced from a currently circulating strain, even if it is antigenically distinct, until an optimally matched and approved vaccine is available. This strategy is to produce a vaccine from an antigenically distant infl uenza (H5N1) strain that could induce broad-spectrum immunity capable of neutralizing newly emerged human H5 strains.

Cell Culture-based Vaccines
Vaccine development based on a cell culture system has advantages over egg-based technology because H5 strains are highly pathogenic for chickens and supplying large numbers of embryonated eggs could be diffi cult in a pandemic. In addition, potential allergic reactions to egg components would be avoided by growing the vaccine virus in tissue culture cells. Recently, mammalian cell culture was used for propagating viruses to prepare killed infl uenza vaccine (28). Inactivated infl uenza vaccines produced with Madin-Darby canine kidney (MDCK) and Vero cells, which served as vaccine substrates, have been licensed in the Netherlands. Of note, the human cell line PER.C6 may provide a useful cell-based system because, unlike MDCK and Vero cell systems, it does not require a solid matrix support for the growth of cells. Selecting background viruses that grow well in these cell cultures and monitoring them for antigenic changes and contaminating microbes during propagation of the virus in cell culture need to be considered.

Development of "Universal" Vaccines
For the development of a universal infl uenza vaccine, a possible target is the relatively conserved M2 homotetramer. The concept is based on identifying alternative infl uenza antigens that are not as susceptible to antigenic shift and drift. Some degree of protection was induced in mice by priming with an M2 ectodomain peptide in adjuvant (29). Studies that used the M2eA peptide conjugated to keyhole limpet hemocyanin and Neisseria meningitidis outer membrane protein illustrated good immune responses not only in mice but also in ferrets and rhesus monkeys (30). In a recent study, 3 M2eA sequences, representing a range of epidemic strains and the (H5N1) strain, were fused to a proprietary hydrophobic protein domain. The resulting fusion proteins, formulated in liposomes, stimulated a protective response in mice challenged with subtypes H1N1, H5N1, H6N2, or H9N2 (31). Previous studies have shown that when M2e is linked to hepatitis B virus core (HBc) particles, it becomes highly immunogenic, eliciting protective antibody response in mice (25). Recently, a series of M2e-HBc constructs were made by increasing the copy number of M2e inserted at the N terminus from 1 to 3 per monomer. The best protection was seen when mice were vaccinated intranasally with these constructs combined with CTA1-DD, a cholera toxin A1-derived mucosal adjuvant (32).
M2 serves as a pH-induced proton channel on the surface of all infl uenza A viruses but is present in low quantities. Further studies are warranted for understanding the mechanism of immune response to M2eA and for defi ning the appropriate immunization conditions for humans.

Vaccination and Correlates of Immune Protection
The lack of established correlates of immunity in animals and humans poses challenges to developing consistent immunologic endpoints for clinical trials and appropriate criteria for vaccine effi cacy. Serum antibody titers, mainly those determined by hemagglutination inhibition (HI) or virus neutralization (VN) assays, or both, are considered surrogate measures of protection. However, the HI test is insensitive for the detection of antibody to avian HA; there also are no recognized clinical correlates of immune protection for neutralization antibody (33,34). Recently, HI or VN assay failed to detect antibodies in ferrets protected by vaccination with whole-virus vaccines containing internal protein from Dk/Sing virus against a heterotypic virus (34). Whether the cross-protection reported is mediated by Tcell response is not known.
In recent years, attempts were made to improve the sensitivity of the HI test. More sensitive detection of anti-body to avian HA was seen when horse erythrocytes were used in place of turkey erythrocytes in the HI test because infl uenza virus was better able to bind to a2,3Gal-specifi c receptor sites on these erythrocytes (35). The presence of asparagines at aa223 (H5 numbering) in H5 HA leads to improved sensitivity of the HI test (22).
Often the immunogenicity of H5 vaccine candidates is assessed by HI or VN assays, but the basis of protection remains unclear. Nevertheless, the tests that are used to evaluate effi cacy of candidate vaccines are based on the assumption that antibody would mediate the protection against infection induced by vaccination, although this has yet to be critically established.
On the basis of initial evidence, infl ammation has been proposed as a possible cause or driving force of avian infl uenza (H5N1). However, components of the infl ammatory response might even be benefi cial. To address these possibilities, we need to determine whether infl ammation in avian infl uenza is an early event and a manifestation of innate immune response. If it is, some of the mediators of innate immune response, such as cytokine/chemokine levels, can be included in the evaluation of the potency of candidate vaccines. Further humoral response as a correlate for protection can be fi ne-tuned by determining the titer and isotype of antibody after vaccination. Several issues concerning vaccine effi cacy are unresolved: What are the consequences of vaccination for existing infl uenza (H5N1) infection, the extent of serologic cross-reactivity between the most closely related types of the virus, and the role in clinical protection? Vaccine administration may provide some therapeutic effects for infected persons who have not yet made an immune response but provide none for those with persistent infection associated with measurable humoral immunity.
Clearly, more studies are warranted to establish a highly reproducible assay to measure immunogenicity of a candidate vaccine and to determine adequate correlates of immune protection. Safety and immunogenicity of adjuvanted vaccines or new formulations should be critically assessed, and any fast-track approval of marketing vaccines must not compromise safety.

Development of Therapeutic (Antidisease) Vaccines
The marked virulence of the 1997 outbreak suggests that infl uenza A (H5N1) infection may have novel pathogenic mechanisms not seen in human infl uenza strains. To attempt to understand pathogenicity of this virus, an infl uenza virus bearing all 8 gene segments of the 1918 pandemic virus, which claimed at least 20-40 million lives, was recently generated in cultured cells. The reconstructed 1918 infl uenza viruses displayed accelerated activation of host immune response in mice with high levels of chemo-kines and cytokines in the lungs, resulting in infi ltration of infl ammatory cells and extensive damage to the lungs with severe hemorrhaging (36). The pathogenicity induced by the reconstructed virus showed marked similarity to that reported with infl uenza (H5N1).
Increasing evidence from mouse models and humans suggests that certain infl ammatory mediators are potent drivers of the disease. If this is true, this could have important implications for developing new therapeutics. Acute respiratory distress syndrome, hemophagocytosis, or both, develop in a substantial fraction of patients with infl uenza (H5N1) infection; both of these conditions are thought to be promoted by overproduction of proinfl ammatory cytokines (known as a "cytokine storm") (37). Consistent with these observations, cytokine release was markedly enhanced in human macrophages after infection with infl uenza (H5N1) (38). Further, marked enhancement of chemokine and cytokine levels was observed in infl uenza (H5N1)-infected persons, particularly in those who died, and these correlated with high and disseminated viral replication (4). Additionally, infl uenza (H5N1) viruses appear relatively resistant to the inhibitory effects of host antiviral cytokines, such as interferons (IFNs) (39). Thus, the severity of human infl uenza (H5N1) infection may be related to the induction of excessive proinfl ammatory responses that can accompany a primary infection and high viral shedding. Increased infl ammation was associated with viral replication in the respiratory and extrarespiratory organs of cats experimentally infected with infl uenza (H5N1) (3). Mice infected with the highly pathogenic infl uenza (H5N1) strain A/HK/156/97, originally obtained from diseased chickens and an ill child in Hong Kong, China (HK), showed reduced ability to activate transforming growth factor-β (TGF-β), a potent antiinfl ammatory cytokine, compared to mice infected with less virulent A/Env/HK437/99 viruses (2). The reduced ability to activate TGF-β may produce greater infl ammation at the site of infection and thus cause more severe disease. Alternatively, the low levels of activated TGF-β in the sera of A/HK/156/97-infected mice may allow the viruses to replicate and spread unchecked in the respiratory tracts of the mice, causing more severe disease. Recently, the impact of the nonstructural (NS) gene variation of Hong Kong (H5N1)/97 on cytokine production was illustrated (40). The NS gene reassortant induced elevated pulmonary concentrations of the infl ammatory cytokines IL-1α, IL-1β, IL-6, IFN-γ, and chemokine KC and decreased concentrations of the anti-infl ammatory cytokine IL-10. This cytokine imbalance is reminiscent of the clinical fi ndings in humans infected with infl uenza (H5N1)/97 virus and may explain the unusual severity of the disease.
The ability to site specifi c engineering changes in the virus genome allows us to consider a novel vaccine approach. By engineering a virus with site-specifi c changes in the genome (for example in NS gene), we may produce infl uenza virus vaccine that favors the production of benefi cial anti-infl ammatory cytokines but remains highly immunogenic. In another approach, a human replicationincompetent, adenoviral vector-based infl uenza vaccine could be developed, in which genes of anti-infl ammatory cytokines are coexpressed, which will inhibit overproduction of proinfl ammatory cytokines. Such vaccines would be considered therapeutic vaccines (e.g., antidisease vaccines), which would inhibit infl ammation at the site of infection and protect against severe disease (Figure). Excessive production of anti-infl ammatory cytokines may result in an inappropriate suppression of the host immune response. Further studies will validate the benefi cial effect of the anti-infl ammatory response for temporizing the cytokine storm seen in infl uenza (H5N1). Development of an immunization protocol that uses an adjuvant that allows selective priming of an antigen-specifi c immunoregulatory cytokines (e.g., IL-10, TGF-β) would be a major advance in the development of a vaccine for bird fl u with a substantial infl ammatory component. The search for potential adjuvants, such as TLR ligands and agonists that will favor the synthesis of inhibitory cytokines including IL-10, should be pursued. By testing whether manipulation of infl ammatory pathways changes the pathologic course, we would identify new targets for disease intervention.

Conclusions
Vaccination is the best option by which to prevent the spread of a pandemic virus and reduce the severity of disease. Defi ning the host response to infl uenza (H5N1) in natural infection is urgently needed to better understand the basis of protection and subsequent development of effi cacious vaccines. Improved vaccine strategies, which will require less antigen and be more robust in inducing both antibody and cell-mediated immunity for neutralizing infl uenza (H5N1) viruses, should be considered. To create an effective vaccine, a combination of factors must be optimized-such as number of doses, formulation without or with better adjuvant, and dose range. We also need to develop a reproducible assay that measures immunogenicity of a vaccine and to establish adequate correlates of protection. The effi cacy of potential cross-reactive vaccine candidates to induce broad-spectrum immunity to infl uenza (H5N1) viruses should be assessed critically; stockpiling of such vaccines may be justifi ed in the absence of optimally matched and approved vaccine during early stages of an H5 pandemic. Search for therapeutic vaccines (antidisease vaccines) aimed at controlling innate immune responses should be pursued, given the clinical evidence that the H5N1 subtype elicits a cytokine storm that contributes to disease pathogenesis. Vaccine development and deploy-ment need to be undertaken by a partnership of academia, government, and industry. The risk for dissemination of pandemic virus will remain if the disease is controlled in 1 area but not in others. A global approach is vital for combating the next infl uenza pandemic, a monumental public health challenge.