Comparative Efficacy of Hemagglutinin,Nucleoprotein, and Matrix 2 Protein Gene-Based Vaccination against H5N1 Influenza in Mouse and Ferret

Efforts to develop a broadly protective vaccine against the highly pathogenic avian influenza A (HPAI) H5N1 virus have focused on highly conserved influenza gene products. The viral nucleoprotein (NP) and ion channel matrix protein (M2) are highly conserved among different strains and various influenza A subtypes. Here, we investigate the relative efficacy of NP and M2 compared to HA in protecting against HPAI H5N1 virus. In mice, previous studies have shown that vaccination with NP and M2 in recombinant DNA and/or adenovirus vectors or with adjuvants confers protection against lethal challenge in the absence of HA. However, we find that the protective efficacy of NP and M2 diminishes as the virulence and dose of the challenge virus are increased. To explore this question in a model relevant to human disease, ferrets were immunized with DNA/rAd5 vaccines encoding NP, M2, HA, NP+M2 or HA+NP+M2. Only HA or HA+NP+M2 vaccination conferred protection against a stringent virus challenge. Therefore, while gene-based vaccination with NP and M2 may provide moderate levels of protection against low challenge doses, it is insufficient to confer protective immunity against high challenge doses of H5N1 in ferrets. These immunogens may require combinatorial vaccination with HA, which confers protection even against very high doses of lethal viral challenge.     

Birth cohort relative to an influenza A virus’s antigenic cluster introduction drives patterns of children’s antibody titers

An individual’s antibody titers to influenza A strains are a result of the complicated interplay between infection history, cross-reactivity, immune waning, and other factors. It has been challenging to disentangle how population-level patterns of humoral immunity change as a function of age, calendar year, and birth cohort from cross-sectional data alone. We analyzed 1,589 longitudinal sera samples from 260 children across three studies in Nicaragua, 2006–16. Hemagglutination inhibition (HAI) titers were determined against four H3N2 strains, one H1N1 strain, and two H1N1pdm strains. We assessed temporal patterns of HAI titers using an age–period–cohort modeling framework. We found that titers against a given virus depended on calendar year of serum collection and birth cohort but not on age. Titer cohort patterns were better described by participants’ ages relative to year of likely introduction of the virus’s antigenic cluster than by age relative to year of strain introduction or by year of birth. These cohort effects may be driven by a decreasing likelihood of early-life infection after cluster introduction and by more broadly reactive antibodies at a young age. H3N2 and H1N1 viruses had qualitatively distinct cohort patterns, with cohort patterns of titers to specific H3N2 strains reaching their peak in children born 3 years prior to that virus’s antigenic cluster introduction and with titers to H1N1 and H1N1pdm strains peaking for children born 1–2 years prior to cluster introduction but not being dramatically lower for older children. Ultimately, specific patterns of strain circulation and antigenic cluster introduction may drive population-level antibody titer patterns in children.     

Single Hemagglutinin Mutations That Alter both Antigenicity and Receptor Binding Avidity Influence Influenza Virus Antigenic Clustering

The hemagglutination inhibition (HAI) assay is the primary measurement used for identifying antigenically novel influenza virus strains. HAI assays measure the amount of reference sera required to prevent virus binding to red blood cells. Receptor binding avidities of viral strains are not usually taken into account when interpreting these assays. Here, we created antigenic maps of human H3N2 viruses that computationally account for variation in viral receptor binding avidities. These new antigenic maps differ qualitatively from conventional antigenic maps based on HAI measurements alone. We experimentally focused on an antigenic cluster associated with a single N145K hemagglutinin (HA) substitution that occurred between 1992 and 1995. Reverse-genetics experiments demonstrated that the N145K HA mutation increases viral receptor binding avidity. Enzyme-linked immunosorbent assays (ELISA) revealed that the N145K HA mutation does not prevent antibody binding; rather, viruses possessing this mutation escape antisera in HAI assays simply by attaching to cells more efficiently. Unexpectedly, we found an asymmetric antigenic effect of the N145K HA mutation. Once H3N2 viruses acquired K145, an epitope involving amino acid 145 became antigenically dominant. Antisera raised against an H3N2 strain possessing K145 had reduced reactivity to H3N2 strains possessing N145. Thus, individual mutations in HA can influence antigenic groupings of strains by altering receptor binding avidity and by changing the dominance of antibody responses. Our results indicate that it will be important to account for variation in viral receptor binding avidity when performing antigenic analyses in order to identify genuine antigenic differences among influenza virus variants.     

Baculovirus Displaying Hemagglutinin Elicits Broad Cross-Protection against Influenza in Mice

The widespread influenza virus infection further emphasizes the need for novel vaccine strategies that effectively reduce the impact of epidemic as well as pandemic influenza. Conventional influenza vaccines generally induce virus neutralizing antibody responses which are specific for a few antigenically related strains within the same subtype. However, antibodies directed against the conserved stalk domain of HA could neutralize multiple subtypes of influenza virus and thus provide broad-spectrum protection. In this study, we designed and constructed a recombinant baculovirus-based vaccine, rBac-HA virus, that expresses full-length HA of pandemic H1N1 influenza virus (A/California/04/09) on the viral envelope. We demonstrated that repeated intranasal immunizations with rBac-HA virus induced HA stalk-specific antibody responses and protective immunity against homologous as well as heterosubtypic virus challenge. The adoptive transfer experiment shows that the cross-protection is conferred by the immune sera which contain HA stalk-specific antibodies. These results warrant further development of rBac-HA virus as a broad-protective vaccine against influenza. The vaccine induced protection against infection with the same subtype as well as different subtype, promising a potential universal vaccine for broad protection against different subtypes to control influenza outbreaks including pandemic.     

Vaccination against Human Influenza A/H3N2 Virus Prevents the Induction of Heterosubtypic Immunity against Lethal Infection with Avian Influenza A/H5N1 Virus

Annual vaccination against seasonal influenza viruses is recommended for certain individuals that have a high risk for complications resulting from infection with these viruses. Recently it was recommended in a number of countries including the USA to vaccinate all healthy children between 6 and 59 months of age as well. However, vaccination of immunologically naïve subjects against seasonal influenza may prevent the induction of heterosubtypic immunity against potentially pandemic strains of an alternative subtype, otherwise induced by infection with the seasonal strains.Here we show in a mouse model that the induction of protective heterosubtypic immunity by infection with a human A/H3N2 influenza virus is prevented by effective vaccination against the A/H3N2 strain. Consequently, vaccinated mice were no longer protected against a lethal infection with an avian A/H5N1 influenza virus. As a result H3N2-vaccinated mice continued to loose body weight after A/H5N1 infection, had 100-fold higher lung virus titers on day 7 post infection and more severe histopathological changes than mice that were not protected by vaccination against A/H3N2 influenza.The lack of protection correlated with reduced virus-specific CD8+ T cell responses after A/H5N1 virus challenge infection. These findings may have implications for the general recommendation to vaccinate all healthy children against seasonal influenza in the light of the current pandemic threat caused by highly pathogenic avian A/H5N1 influenza viruses.     

Transient Humoral Protection against H5N1 Challenge after Seasonal Influenza Vaccination of Humans

Current influenza vaccines are believed to confer protection against a narrow range of virus strains. The identification of broadly influenza neutralizing antibodies (bnAbs) has triggered efforts to develop vaccines providing ‘universal’ protection against influenza. Several bnAbs were isolated from humans recently vaccinated with conventional influenza vaccines, suggesting that such vaccines could, in principle, be broadly protective. Assessing the breadth-of-protection conferred to humans by influenza vaccines is hampered by the lack of in vitro correlates for broad protection. We designed and employed a novel human-to-mouse serum transfer and challenge model to analyze protective responses in serum samples from clinical trial subjects. One dose of seasonal vaccine induces humoral protection not only against vaccine-homologous H1N1 challenge, but also against H5N1 challenge. This heterosubtypic protection is neither detected, nor accurately predicted by in vitro immunogenicity assays. Moreover, heterosubtypic protection is transient and not boosted by repeated inoculations. Strategies to increase the breadth and duration of the protective response against influenza are required to obtain ‘universal’ protection against influenza by vaccination. In the absence of known correlates of protection for broadly protective vaccines, the human-to-mouse serum transfer and challenge model described here may aid the development of such vaccines.     

Induction of heterosubtypic immunity to influenza virus by intranasal immunization

Recovery from live influenza virus infection is known to induce heterosubtypic immunity. In contrast, immunity induced by inactivated vaccines is predominantly subtype specific. In this study, we investigated the heterosubtypic protective immunity induced by inactivated influenza virus. Intranasal immunization of mice with inactivated influenza virus A/PR8 (H1N1) provided complete protection against the homologous virus and a drift virus within the same subtype, A/WSN (H1N1), but not against the heterosubtypic virus A/Philippines (H3N2). However, coadministration of inactivated virus with cholera toxin as an adjuvant conferred complete heterosubtypic protection, without observed illness, even under conditions of CD4⁺ or CD8⁺ T-cell depletion. Analysis of immune correlates prior to challenge and postchallenge indicated that humoral immune responses with cross-neutralizing activity in lungs and in sera play a major role in conferring protective immunity against heterosubtypic challenge. This study has significant implications for developing broadly cross-reactive vaccines against newly emerging pathogenic influenza viruses.     

A novel role for non-neutralizing antibodies against nucleoprotein in facilitating resistance to influenza virus

Current influenza vaccines elicit Abs to the hemagglutinin and neuraminidase envelope proteins. Due to antigenic drift, these vaccines must be reformulated annually to include the envelope proteins predicted to dominate in the following season. By contrast, vaccination with the conserved nucleoprotein (NP) elicits immunity against multiple serotypes (heterosubtypic immunity). NP vaccination is generally thought to convey protection primarily via CD8 effector mechanisms. However, significant titers of anti-NP Abs are also induced, yet the involvement of Abs in protection has largely been disregarded. To investigate how Ab responses might contribute to heterosubtypic immunity, we vaccinated C57BL/6 mice with soluble rNP. This approach induced high titers of NP-specific serum Ab, but only poorly detectable NP-specific T cell responses. Nevertheless, rNP immunization significantly reduced morbidity and viral titers after influenza challenge. Importantly, Ab-deficient mice were not protected by this vaccination strategy. Furthermore, rNP-immune serum could transfer protection to naive hosts in an Ab-dependent manner. Therefore, Ab to conserved, internal viral proteins, such as NP, provides an unexpected, yet important mechanism of protection against influenza. These results suggest that vaccines designed to elicit optimal heterosubtypic immunity to influenza should promote both Ab and T cell responses to conserved internal proteins.     

Design of a heterosubtypic epitope-based peptide vaccine fused with hemokinin-1 against influenza viruses

Influenza viruses continue to emerge and re-emerge, posing new threats for public health. Control and treatment of influenza depends mainly on vaccination and chemoprophylaxis with approved antiviral drugs. Identification of specific epitopes derived from influenza viruses has significantly advanced the development of epitope-based vaccines. Here, we explore the idea of using HLA binding data to design an epitope-based vaccine that can elicit heterosubtypic T-cell responses against circulating H7N9, H5N1, and H9N2 subtypes. The hemokinin-1(HK-1) peptide sequence was used to induce immune responses against the influenza viruses. Five conserved high score cytotoxic T lymphocyte(CTL) epitopes restricted to HLA-A*0201-binding peptides within the hemagglutinin(HA) protein of the viruses were chosen, and two HA CTL/HK-1 chimera protein models designed. Using in silico analysis, which involves interferon epitope scanning, protein structure prediction, antigenic epitope determination, and model quality evaluation, chimeric proteins were designed. The applicability of one of these proteins as a heterosubtypic epitopebased vaccine candidate was analyzed.     

Heterosubtypic Immunity to Influenza A Virus Infections in Mallards May Explain Existence of Multiple Virus Subtypes

Wild birds, particularly duck species, are the main reservoir of influenza A virus (IAV) in nature. However, knowledge of IAV infection dynamics in the wild bird reservoir, and the development of immune responses, are essentially absent. Importantly, a detailed understanding of how subtype diversity is generated and maintained is lacking. To address this, 18,679 samples from 7728 Mallard ducks captured between 2002 and 2009 at a single stopover site in Sweden were screened for IAV infections, and the resulting 1081 virus isolates were analyzed for patterns of immunity. We found support for development of homosubtypic hemagglutinin (HA) immunity during the peak of IAV infections in the fall. Moreover, re-infections with the same HA subtype and related prevalent HA subtypes were uncommon, suggesting the development of natural homosubtypic and heterosubtypic immunity (p-value = 0.02). Heterosubtypic immunity followed phylogenetic relatedness of HA subtypes, both at the level of HA clades (p-value = 0.04) and the level of HA groups (p-value = 0.05). In contrast, infection patterns did not support specific immunity for neuraminidase (NA) subtypes. For the H1 and H3 Clades, heterosubtypic immunity showed a clear temporal pattern and we estimated within-clade immunity to last at least 30 days. The strength and duration of heterosubtypic immunity has important implications for transmission dynamics of IAV in the natural reservoir, where immune escape and disruptive selection may increase HA antigenic variation and explain IAV subtype diversity.     

Masking of antigenic epitopes by antibodies shapes the humoral immune response to influenza

The immune responses to influenza, a virus that exhibits strain variation, show complex dynamics where prior immunity shapes the response to the subsequent infecting strains. Original antigenic sin (OAS) describes the observation that antibodies to the first encountered influenza strain, specifically antibodies to the epitopes on the head of influenza''s main surface glycoprotein, haemagglutinin (HA), dominate following infection with new drifted strains. OAS suggests that responses to the original strain are preferentially boosted. Recent studies also show limited boosting of the antibodies to conserved epitopes on the stem of HA, which are attractive targets for a ‘universal vaccine’. We develop multi-epitope models to explore how pre-existing immunity modulates the immune response to new strains following immunization. Our models suggest that the masking of antigenic epitopes by antibodies may play an important role in describing the complex dynamics of OAS and limited boosting of antibodies to the stem of HA. Analysis of recently published data confirms model predictions for how pre-existing antibodies to an epitope on HA decrease the magnitude of boosting of the antibody response to this epitope following immunization. We explore strategies for boosting of antibodies to conserved epitopes and generating broadly protective immunity to multiple strains.     

A Trivalent Virus-Like Particle Vaccine Elicits Protective Immune Responses against Seasonal Influenza Strains in Mice and Ferrets

There is need for improved human influenza vaccines, particularly for older adults who are at greatest risk for severe disease, as well as to address the continuous antigenic drift within circulating human subtypes of influenza virus. We have engineered an influenza virus-like particle (VLP) as a new generation vaccine candidate purified from the supernatants of Sf9 insect cells following infection by recombinant baculoviruses to express three influenza virus proteins, hemagglutinin (HA), neuraminidase (NA), and matrix 1 (M1). In this study, a seasonal trivalent VLP vaccine (TVV) formulation, composed of influenza A H1N1 and H3N2 and influenza B VLPs, was evaluated in mice and ferrets for the ability to elicit antigen-specific immune responses. Animals vaccinated with the TVV formulation had hemagglutination-inhibition (HAI) antibody titers against all three homologous influenza virus strains, as well as HAI antibodies against a panel of heterologous influenza viruses. HAI titers elicited by the TVV were statistically similar to HAI titers elicited in animals vaccinated with the corresponding monovalent VLP. Mice vaccinated with the TVV had higher level of influenza specific CD8+ T cell responses than a commercial trivalent inactivated vaccine (TIV). Ferrets vaccinated with the highest dose of the VLP vaccine and then challenged with the homologous H3N2 virus had the lowest titers of replicating virus in nasal washes and showed no signs of disease. Overall, a trivalent VLP vaccine elicits a broad array of immunity and can protect against influenza virus challenge.     

The Role of Social Contacts and Original Antigenic Sin in Shaping the Age Pattern of Immunity to Seasonal Influenza

Recent serological studies of seasonal influenza A in humans suggest a striking characteristic profile of immunity against age, which holds across different countries and against different subtypes of influenza. For both H1N1 and H3N2, the proportion of the population seropositive to recently circulated strains peaks in school-age children, reaches a minimum between ages 35–65, then rises again in the older ages. This pattern is little understood. Variable mixing between different age classes can have a profound effect on disease dynamics, and is hence the obvious candidate explanation for the profile, but using a mathematical model of multiple influenza strains, we see that age dependent transmission based on mixing data from social contact surveys cannot on its own explain the observed pattern. Instead, the number of seropositive individuals in a population may be a consequence of ‘original antigenic sin’; if the first infection of a lifetime dominates subsequent immune responses, we demonstrate that it is possible to reproduce the observed relationship between age and seroprevalence. We propose a candidate mechanism for this relationship, by which original antigenic sin, along with antigenic drift and vaccination, results in the age profile of immunity seen in empirical studies.     

Pre-existing heterosubtypic immunity provides a barrier to airborne transmission of influenza viruses

Human-to-human transmission of influenza viruses is a serious public health threat, yet the precise role of immunity from previous infections on the susceptibility to airborne infection is still unknown. Using the ferret model, we examined the roles of exposure duration and heterosubtypic immunity on influenza transmission. We demonstrate that a 48 hour exposure is sufficient for efficient transmission of H1N1 and H3N2 viruses. To test pre-existing immunity, a gap of 8–12 weeks between primary and secondary infections was imposed to reduce innate responses and ensure robust infection of donor animals with heterosubtypic viruses. We found that pre-existing H3N2 immunity did not significantly block transmission of the 2009 H1N1pandemic (H1N1pdm09) virus to immune animals. Surprisingly, airborne transmission of seasonal H3N2 influenza strains was abrogated in recipient animals with H1N1pdm09 pre-existing immunity. This protection from natural infection with H3N2 virus was independent of neutralizing antibodies. Pre-existing immunity with influenza B virus did not block H3N2 virus transmission, indicating that the protection was likely driven by the adaptive immune response. We demonstrate that pre-existing immunity can impact susceptibility to heterologous influenza virus strains, and implicate a novel correlate of protection that can limit the spread of respiratory pathogens through the air.     

Cooperativity Between CD8+ T Cells,Non-Neutralizing Antibodies,and Alveolar Macrophages Is Important for Heterosubtypic Influenza Virus Immunity

Seasonal epidemics of influenza virus result in ∼36,000 deaths annually in the United States. Current vaccines against influenza virus elicit an antibody response specific for the envelope glycoproteins. However, high mutation rates result in the emergence of new viral serotypes, which elude neutralization by preexisting antibodies. T lymphocytes have been reported to be capable of mediating heterosubtypic protection through recognition of internal, more conserved, influenza virus proteins. Here, we demonstrate using a recombinant influenza virus expressing the LCMV GP33-41 epitope that influenza virus-specific CD8+ T cells and virus-specific non-neutralizing antibodies each are relatively ineffective at conferring heterosubtypic protective immunity alone. However, when combined virus-specific CD8 T cells and non-neutralizing antibodies cooperatively elicit robust protective immunity. This synergistic improvement in protective immunity is dependent, at least in part, on alveolar macrophages and/or other lung phagocytes. Overall, our studies suggest that an influenza vaccine capable of eliciting both CD8+ T cells and antibodies specific for highly conserved influenza proteins may be able to provide heterosubtypic protection in humans, and act as the basis for a potential “universal” vaccine.     

Annual vaccination against influenza virus hampers development of virus-specific CD8⁺ T cell immunity in children

Infection with seasonal influenza A viruses induces immunity to potentially pandemic influenza A viruses of other subtypes (heterosubtypic immunity). We recently demonstrated that vaccination against seasonal influenza prevented the induction of heterosubtypic immunity against influenza A/H5N1 virus induced by infection with seasonal influenza in animal models, which correlated with the absence of virus-specific CD8(+) T cell responses. Annual vaccination of all healthy children against influenza has been recommended, but the impact of vaccination on the development of the virus-specific CD8(+) T cell immunity in children is currently unknown. Here we compared the virus-specific CD8(+) T cell immunity in children vaccinated annually with that in unvaccinated children. In the present study, we compared influenza A virus-specific cellular and humoral responses of unvaccinated healthy control children with those of children with cystic fibrosis (CF) who were vaccinated annually. Similar virus-specific CD4(+) T cell and antibody responses were observed, while an age-dependent increase of the virus-specific CD8(+) T cell response that was absent in vaccinated CF children was observed in unvaccinated healthy control children. Our results indicate that annual influenza vaccination is effective against seasonal influenza but hampers the development of virus-specific CD8(+) T cell responses. The consequences of these findings are discussed in the light of the development of protective immunity to seasonal and future pandemic influenza viruses.     

Estimating the Life Course of Influenza A(H3N2) Antibody Responses from Cross-Sectional Data

The immunity of a host population against specific influenza A strains can influence a number of important biological processes, from the emergence of new virus strains to the effectiveness of vaccination programmes. However, the development of an individual’s long-lived antibody response to influenza A over the course of a lifetime remains poorly understood. Accurately describing this immunological process requires a fundamental understanding of how the mechanisms of boosting and cross-reactivity respond to repeated infections. Establishing the contribution of such mechanisms to antibody titres remains challenging because the aggregate effect of immune responses over a lifetime are rarely observed directly. To uncover the aggregate effect of multiple influenza infections, we developed a mechanistic model capturing both past infections and subsequent antibody responses. We estimated parameters of the model using cross-sectional antibody titres to nine different strains spanning 40 years of circulation of influenza A(H3N2) in southern China. We found that “antigenic seniority” and quickly decaying cross-reactivity were important components of the immune response, suggesting that the order in which individuals were infected with influenza strains shaped observed neutralisation titres to a particular virus. We also obtained estimates of the frequency and age distribution of influenza infection, which indicate that although infections became less frequent as individuals progressed through childhood and young adulthood, they occurred at similar rates for individuals above age 30 y. By establishing what are likely to be important mechanisms driving epochal trends in population immunity, we also identified key directions for future studies. In particular, our results highlight the need for longitudinal samples that are tested against multiple historical strains. This could lead to a better understanding of how, over the course of a lifetime, fast, transient antibody dynamics combine with the longer-term immune responses considered here.     

Vaccination against seasonal influenza A/H3N2 virus reduces the induction of heterosubtypic immunity against influenza A/H5N1 virus infection in ferrets

Infection with seasonal influenza viruses induces a certain extent of protective immunity against potentially pandemic viruses of novel subtypes, also known as heterosubtypic immunity. Here we demonstrate that infection with a recent influenza A/H3N2 virus strain induces robust protection in ferrets against infection with a highly pathogenic avian influenza virus of the H5N1 subtype. Prior H3N2 virus infection reduced H5N1 virus replication in the upper respiratory tract, as well as clinical signs, mortality, and histopathological changes associated with virus replication in the brain. This protective immunity correlated with the induction of T cells that cross-reacted with H5N1 viral antigen. We also demonstrated that prior vaccination against influenza A/H3N2 virus reduced the induction of heterosubtypic immunity otherwise induced by infection with the influenza A/H3N2 virus. The implications of these findings are discussed in the context of vaccination strategies and vaccine development aiming at the induction of immunity to pandemic influenza.     

Priming with cold-adapted influenza A does not prevent infection but elicits long-lived protection against supralethal challenge with heterosubtypic virus

We show in this study several novel features of T cell-based heterosubtypic immunity against the influenza A virus in mice. First, T cell-mediated heterosubtypic protection against lethal challenge can be generated by a very low priming dose. Second, it becomes effective within 5-6 days. Third, it provides protection against a very high dose challenge for >70 days. Also novel is the finding that strong, long-lasting, heterosubtypic protection can be elicited by priming with attenuated cold-adapted strains. We demonstrate that priming does not prevent infection of the lungs following challenge, but leads to earlier clearance of the virus and 100% survival after otherwise lethal challenge. Protection is dependent on CD8 T cells, and we show that CD4 and CD8 T cells reactive to conserved epitopes of the core proteins of the challenge virus are present after priming. Our results suggest that intranasal vaccination with cold-adapted, attenuated live virus has the potential to provide effective emergency protection against emerging influenza strains for several months.     

Antigenic and genetic evolution of equine influenza A (H3N8) virus from 1968 to 2007

Equine influenza virus is a major respiratory pathogen in horses, and outbreaks of disease often lead to substantial disruption to and economic losses for equestrian industries. The hemagglutinin (HA) protein is of key importance in the control of equine influenza because HA is the primary target of the protective immune response and the main component of currently licensed influenza vaccines. However, the influenza virus HA protein changes over time, a process called antigenic drift, and vaccine strains must be updated to remain effective. Antigenic drift is assessed primarily by the hemagglutination inhibition (HI) assay. We have generated HI assay data for equine influenza A (H3N8) viruses isolated between 1968 and 2007 and have used antigenic cartography to quantify antigenic differences among the isolates. The antigenic evolution of equine influenza viruses during this period was clustered: from 1968 to 1988, all isolates formed a single antigenic cluster, which then split into two cocirculating clusters in 1989, and then a third cocirculating cluster appeared in 2003. Viruses from all three clusters were isolated in 2007. In one of the three clusters, we show evidence of antigenic drift away from the vaccine strain over time. We determined that a single amino acid substitution was likely responsible for the antigenic differences among clusters.