Drift Variants Research Articles

Sequential Infection in Ferrets with Antigenically Distinct Seasonal H1N1 Influenza Viruses Boosts Hemagglutinin Stalk-Specific Antibodies.

Broadly reactive antibodies targeting the conserved hemagglutinin (HA) stalk region are elicited following sequential infection or vaccination with influenza viruses belonging to divergent subtypes and/or expressing antigenically distinct HA globular head domains. Here, we demonstrate, through the use of novel chimeric HA proteins and competitive binding assays, that sequential infection of ferrets with antigenically distinct seasonal H1N1 (sH1N1) influenza virus isolates induced an HA stalk-specific antibody response. Additionally, stalk-specific antibody titers were boosted following sequential infection with antigenically distinct sH1N1 isolates in spite of preexisting, cross-reactive, HA-specific antibody titers. Despite a decline in stalk-specific serum antibody titers, sequential sH1N1 influenza virus-infected ferrets were protected from challenge with a novel H1N1 influenza virus (A/California/07/2009), and these ferrets poorly transmitted the virus to naive contacts. Collectively, these findings indicate that HA stalk-specific antibodies are commonly elicited in ferrets following sequential infection with antigenically distinct sH1N1 influenza virus isolates lacking HA receptor-binding site cross-reactivity and can protect ferrets against a pathogenic novel H1N1 virus. The influenza virus hemagglutinin (HA) is a major target of the humoral immune response following infection and/or seasonal vaccination. While antibodies targeting the receptor-binding pocket of HA possess strong neutralization capacities, these antibodies are largely strain specific and do not confer protection against antigenic drift variant or novel HA subtype-expressing viruses. In contrast, antibodies targeting the conserved stalk region of HA exhibit broader reactivity among viruses within and among influenza virus subtypes. Here, we show that sequential infection of ferrets with antigenically distinct seasonal H1N1 influenza viruses boosts the antibody responses directed at the HA stalk region. Moreover, ferrets possessing HA stalk-specific antibody were protected against novel H1N1 virus infection and did not transmit the virus to naive contacts.

In-vitro assessment of differential cytokine gene expression in response to infections with Egyptian classic and variant strains of highly pathogenic H5N1 avian influenza virus

In Egypt, two distinct genetic groups of HPAI H5N1 viruses are co-circulating: classic 2.2.1/C sub-clade and antigenic drift variant 2.2.1.1 clade isolated from vaccinated poultry flocks. The response of chicken innate immunity to both genotypes is not investigated, so far. In this study, expression of immune related genes (IL1b, IL4, IL6, IL8, IL10, IL18, IFNα and IFNγ) after infecting chicken macrophage cell line (HD11) and chicken peripheral blood Mononuclear cells (PBMC) with a classic and a variant strains was assayed using quantitative reverse-transcription real-time polymerase chain reaction assays (qRT-PCR). In HD11, the variant strain induced higher levels of IL1b and IL8 at 6hours post infection (hpi), IL4 at 24 / 48hpi and IFNα at 48hpi than the classic strain. Conversely, the classic strain induced about 10-fold increase of IFNγ at 24 and 48hpi and the virus replicated at higher level than the variant strain. The results of PBMC infection were similar to that reported from HD11 except for IFNγ gene expression that was higher at variant strain infected cells than that infected with the classic strain. After 24hpi skewing the innate immune response toward anti-inflammatory (humoral-associated) cytokines was different between HD11 (through IL4) and PBMC (through IL10). To sum up, the classic strain produced less cytokines which may indicate adaptation to evade the recognition by the innate immune system and explain its higher pathogenicity.

Programming of Influenza Vaccine Broadness and Persistence by Mucoadhesive Polymer-Based Adjuvant Systems.

The development of an anti-influenza vaccine with the potential for cross-protection against seasonal drift variants as well as occasionally emerging reassortant viruses is essential. In this study, we successfully generated a novel anti-influenza vaccine system combining conserved matrix protein 2 (sM2) and stalk domain of hemagglutinin (HA2) fusion protein (sM2HA2) and poly-γ-glutamic acid (γ-PGA)-based vaccine adjuvant systems that can act as a mucoadhesive delivery vehicle of sM2HA2 as well as a robust strategy for the incorporation of hydrophobic immunostimulatory 3-O-desacyl-4'-monophosphoryl lipid A (MPL) and QS21. Intranasal coadministration of sM2HA2 and the combination adjuvant γ-PGA/MPL/QS21 (CA-PMQ) was able to induce a high degree of protective mucosal, systemic, and cell-mediated immune responses. The sM2HA2/CA-PMQ immunization was able to prevent disease symptoms, confering complete protection against lethal infection with divergent influenza subtypes (H5N1, H1N1, H5N2, H7N3, and H9N2) that lasted for at least 6 mo. Therefore, our data suggest that mucosal administration of sM2HA2 in combination with CA-PMQ could be a potent strategy for a broad cross-protective influenza vaccine, and CA-PMQ as a mucosal adjuvant could be used for effective mucosal vaccines.

Evolution of the hemagglutinin expressed by human influenza A(H1N1)pdm09 and A(H3N2) viruses circulating between 2008–2009 and 2013–2014 in Germany

This report describes the evolution of the influenza A(H1N1)pdm09 and A(H3N2) viruses circulating in Germany between 2008–2009 and 2013–2014. The phylogenetic analysis of the hemagglutinin (HA) genes of both subtypes revealed similar evolution of the HA variants that were also seen worldwide with minor exceptions. The analysis showed seven distinct HA clades for A(H1N1)pdm09 and six HA clades for A(H3N2) viruses. Herald strains of both subtypes appeared sporadically since 2008–2009. Regarding A(H1N1)pdm09, herald strains of HA clade 3 and 4 were detected late in the 2009–2010 season. With respect to A(H3N2), we found herald strains of HA clade 3, 4 and 7 between 2009 and 2012. Those herald strains were predominantly seen for minor and not for major HA clades. Generally, amino acid substitutions were most frequently found in the globular domain, including substitutions near the antigenic sites or the receptor binding site. Differences between both influenza A subtypes were seen with respect to the position of the indicated substitutions in the HA. For A(H1N1)pdm09 viruses, we found more substitutions in the stem region than in the antigenic sites. In contrast, in A(H3N2) viruses most changes were identified in the major antigenic sites and five changes of potential glycosylation sites were identified in the head of the HA monomer. Interestingly, we found in seasons with less influenza activity a relatively high increase of substitutions in the head of the HA in both subtypes. This might be explained by the fact that mutations under negative selection are subsequently compensated by secondary mutations to restore important functions e.g. receptor binding properties. A better knowledge of basic evolution strategies of influenza viruses will contribute to the refinement of predictive mathematical models for identifying novel antigenic drift variants.

Focused antibody response to influenza linked to antigenic drift.

The selective pressure that drives antigenic changes in influenza viruses is thought to originate from the human immune response. Here, we have characterized the B cell repertoire from a previously vaccinated donor whose serum had reduced neutralizing activity against the recently evolved clade 6B H1N1pdm09 viruses. While the response was markedly polyclonal, 88% of clones failed to recognize clade 6B viruses; however, the ability to neutralize A/USSR/90/1977 influenza, to which the donor would have been exposed in childhood, was retained. In vitro selection of virus variants with representative monoclonal antibodies revealed that a single amino acid replacement at residue K163 in the Sa antigenic site, which is characteristic of the clade 6B viruses, was responsible for resistance to neutralization by multiple monoclonal antibodies and the donor serum. The K163 residue lies in a part of a conserved surface that is common to the hemagglutinins of the 1977 and 2009 H1N1 viruses. Vaccination with the 2009 hemagglutinin induced an antibody response tightly focused on this common surface that is capable of selecting current antigenic drift variants in H1N1pdm09 influenza viruses. Moreover, amino acid replacement at K163 was not highlighted by standard ferret antisera. Human monoclonal antibodies may be a useful adjunct to ferret antisera for detecting antigenic drift in influenza viruses.

Haemagglutinin mutations and glycosylation changes shaped the 2012/13 influenza A(H3N2) epidemic, Houston, Texas.

While the early start and higher intensity of the 2012/13 influenza A virus (IAV) epidemic was not unprecedented, it was the first IAV epidemic season since the 2009 H1N1 influenza pandemic where the H3N2 subtype predominated. We directly sequenced the genomes of 154 H3N2 clinical specimens collected throughout the epidemic to better understand the evolution of H3N2 strains and to inform the H3N2 vaccine selection process. Phylogenetic analyses indicated that multiple co-circulating clades and continual antigenic drift in the haemagglutinin (HA) of clades 5, 3A, and 3C, with the evolution of a new 3C subgroup (3C-2012/13), were the driving causes of the epidemic. Drift variants contained HA substitutions and alterations in the potential N-linked glycosylation sites of HA. Antigenic analysis demonstrated that viruses in the emerging subclade 3C.3 and subgroup 3C-2012/13 were not well inhibited by antisera generated against the 3C.1 vaccine strains used for the 2012/13 (A/Victoria/361/2011) or 2013/14 (A/Texas/50/2012) seasons. Our data support updating the H3N2 vaccine strain to a clade 3C.2 or 3C.3-like strain or a subclade that has drifted further. They also underscore the challenges in vaccine strain selection, particularly regarding HA and neuraminidase substitutions derived during laboratory passage that may alter antigenic testing accuracy.

Influenza B virus-specific CD8+ T-lymphocytes strongly cross-react with viruses of the opposing influenza B lineage.

Influenza B viruses fall in two antigenically distinct lineages (B/Victoria/2/1987 and B/Yamagata/16/1988 lineage) that co-circulate with influenza A viruses of the H3N2 and H1N1 subtypes during seasonal epidemics. Infections with influenza B viruses contribute considerably to morbidity and mortality in the human population. Influenza B virus neutralizing antibodies, elicited by natural infections or vaccination, poorly cross-react with viruses of the opposing influenza B lineage. Therefore, there is an increased interest in identifying other correlates of protection which could aid the development of broadly protective vaccines. blast analysis revealed high sequence identity of all viral proteins. With two online epitope prediction algorithms, putative conserved epitopes relevant for study subjects used in the present study were predicted. The cross-reactivity of influenza B virus-specific polyclonal CD8+ cytotoxic T-lymphocyte (CTL) populations obtained from HLA-typed healthy study subjects, with intra-lineage drift variants and viruses of the opposing lineage, was determined by assessing their in vitro IFN-γ response and lytic activity. Here, we show for the first time, to the best of our knowledge, that CTLs directed to viruses of the B/Victoria/2/1987 lineage cross-react with viruses of the B/Yamagata/16/1988 lineage and vice versa.

Developing Universal Influenza Vaccines: Hitting the Nail, Not Just on the Head.

Influenza viruses have a huge impact on public health. Current influenza vaccines need to be updated annually and protect poorly against antigenic drift variants or novel emerging subtypes. Vaccination against influenza can be improved in two important ways, either by inducing more broadly protective immune responses or by decreasing the time of vaccine production, which is relevant especially during a pandemic outbreak. In this review, we outline the current efforts to develop so-called “universal influenza vaccines”, describing antigens that may induce broadly protective immunity and novel vaccine production platforms that facilitate timely availability of vaccines.

Use of Computational Matrix Adjustment to Evaluate the Effectiveness of Common Influenza Vaccines against the Emergence of Drift Variants

Every year the FDA issues a recommendation for the composition of the year’s common influenza vaccine for influenzas A and B. The FDA can consistently predict the dominance of a particular strand of influenza virus by taking into account previous years’ antigenic characterization percentages. However, the sudden disappearance of dominant antigens and the sudden emergence of drift variants can disrupt this pattern, which questions the effectiveness of that year’s vaccine. Basic Local Alignment Search Tool was used to compare the protein sequences for hemagglutinin and neuraminidase between the strands in the vaccine and the dominant viral strands. This study examined the effectiveness of vaccines from 2000 to 2012, focusing on the transitions between the B/Yamagata and B/Victoria lineages and A/New Caledonia and A/California lineages (H1N1). Between the years 2005 and 2006, dominance of the B/Yamagata lineage, represented by B/Shanghai/361/2002, disappeared almost entirely. For the 2005-2006 flu season, the CDC recommended a B/Shanghai/361/2002 vaccine which expressed a 98% identity to the dominant influenza B hemagglutinin sequence and a 97% identity to the dominant neuraminidase sequence. From 2007 to 2008, the A/New Caledonia virus declined to 34% of cases while the A/Solomon Islands/3/2006 virus increased to 66%. The A/New Caledonia/20/99 vaccine effectively expressed a 97% identity to the hemagglutinin sequence of A/Solomon Islands/3/2006 strand and a 98% identity to the neuraminidase sequence. This study demonstrates that from 2000 to 2012, despite drift variants in influenza viruses, the CDC-recommended vaccine effectively matches the hemagglutinin and neuraminidase protein sequences of the dominant viruses.DOI: http://dx.doi.org/10.3126/ijasbt.v2i3.10952 Int J Appl Sci Biotechnol, Vol. 2(3): 224-228

Committing the Oldest Sins in the Newest Kind of Ways-Antibodies Targeting the Influenza Virus Type A Hemagglutinin Globular Head.

The globular head of the trimeric influenza hemagglutinin (HA) contains the receptor-binding domain (RBD) and is the target of potently neutralizing human monoclonal antibodies (mAbs) with high in vivo activity. In general, these mAbs are induced easily by vaccination, but only infrequently display cross-neutralizing activity against antigenic drift variants or even against HA molecules from viruses of heterologous subtypes. Recently, the atomic resolution structures of several such antibodies in complex with HA have been determined by X-ray crystallography. Not surprisingly, cross-reactive globular head antibodies target, at least partially, the conserved RBD. The cross-reactive potential of such mAbs is limited by contacts of hypervariable HA residues outside the conserved RBD. The RBD of H2 HA seems especially immunogenic. Increasing the immunogenicity of the RBD of other HA subtypes may be a step toward a universal influenza vaccine. The germ line-encoded Phe54 residue of the CDR-H2 of the VH1-69 germ line sequence appears to be ideally suited not only to reach into a conserved, hydrophobic pocket on the HA stem, but also to reach into the conserved, hydrophobic pocket that is the RBD. We have cloned antibodies from different individuals that are encoded by the VH1-69 germ line gene segment that contact the universally conserved Trp153 on the bottom of the RBD. These antibodies serve as further evidence of antibody genetic sequence convergence across individuals.

4Flu - an individual based simulation tool to study the effects of quadrivalent vaccination on seasonal influenza in Germany.

BackgroundInfluenza vaccines contain Influenza A and B antigens and are adjusted annually to match the characteristics of circulating viruses. In Germany, Influenza B viruses belonged to the B/Yamagata lineage, but since 2001, the antigenically distinct B/Victoria lineage has been co-circulating. Trivalent influenza vaccines (TIV) contain antigens of the two A subtypes A(H3N2) and A(H1N1), yet of only one B lineage, resulting in frequent vaccine mismatches. Since 2012, the WHO has been recommending vaccine strains from both B lineages, paving the way for quadrivalent influenza vaccines (QIV).MethodsUsing an individual-based simulation tool, we simulate the concomitant transmission of four influenza strains, and compare the effects of TIV and QIV on the infection incidence. Individuals are connected in a dynamically evolving age-dependent contact network based on the POLYMOD matrix; their age-distribution reproduces German demographic data and predictions. The model considers maternal protection, boosting of existing immunity, loss of immunity, and cross-immunizing events between the B lineages. Calibration to the observed annual infection incidence of 10.6% among young adults yielded a basic reproduction number of 1.575. Vaccinations are performed annually in October and November, whereby coverage depends on the vaccinees’ age, their risk status and previous vaccination status. New drift variants are introduced at random time points, leading to a sudden loss of protective immunity for part of the population and occasionally to reduced vaccine efficacy. Simulations run for 50 years, the first 30 of which are used for initialization. During the final 20 years, individuals receive TIV or QIV, using a mirrored simulation approach.ResultsUsing QIV, the mean annual infection incidence can be reduced from 8,943,000 to 8,548,000, i.e. by 395,000 infections, preventing 11.2% of all Influenza B infections which still occur with TIV (95% CI: 10.7-11.8%). Using a lower B lineage cross protection than the baseline 60%, the number of Influenza B infections increases and the number additionally prevented by QIV can be 5.5 times as high.ConclusionsVaccination with TIV substantially reduces the Influenza incidence compared to no vaccination. Depending on the assumed degree of B lineage cross protection, QIV further reduces Influenza B incidence by 11-33%.

Chimerization and characterization of a monoclonal antibody with potent neutralizing activity across multiple influenza A H5N1 clades

The persistent evolution and circulation of highly pathogenic avian influenza H5N1 viruses pose a serious threat to global heath and hamper pandemic preparedness through conventional vaccine strategies. Combination passive immunotherapy using non-competing neutralizing antibodies has been proposed as a viable alternative to provide broad protection against drift variants. This necessitates the pre-pandemic production and characterization of potently neutralizing monoclonal antibodies (MAbs). One such antibody, MAb 9F4 was shown to provide heterologous protection against multiple H5N1 clade viruses, including one of the recently designated subclades, namely 2.3.4, through binding to a novel epitope, warranting its further development and characterization as a therapeutic candidate. In this study, the conversion of MAb 9F4 from mouse IgG2b to mouse-human chimeric (xi) IgG1 and IgA1 was achieved. These chimeric MAb versions were found to retain high degrees of binding and neutralizing activity against H5N1. The demonstration that xi-IgA1-9F4 retains a fairly high level of neutralizing activity, which is ∼10-fold lower than the corresponding xi-IgG1 isotype, suggests that this MAb could be further developed and engineered for intranasal administration.

Population-Based Hospitalization Burden of Influenza A Virus Subtypes and Antigenic Drift Variants in Children in Hong Kong (2004–2011)

ObjectivesWe aim to document and analyze influenza hospitalization burden in light of antigenic changes in circulating influenza viruses in Hong Kong.MethodsThe pediatric age-specific rates of influenza A hospitalization in Hong Kong for 2004–2011 which encompassed the emergence of H1N1pdm09 were extrapolated from admissions to 2 hospitals that together catered for 72.5% of all pediatric admissions on Hong Kong Island. Influenza A was detected by immunofluorescence, culture and/or PCR on nasopharyngeal aspirates.ResultsInfluenza A caused high rates of hospitalization in children with year to year fluctuations. The highest hospitalization burden was seen with H1N1pdm09 in 2009. Additional factors affecting hospitalization were the proportion of viral circulation among different subtypes, and antigenic drifts. Taking these into effect, an H3N2 dominated year was not always associated with more hospitalizations than a ‘seasonal’ H1N1 year. Hospitalization burden was higher in seasons when drifted viruses of H1N1 or H3N2 dominated. No hospitalization was documented in infants <6 months of age during years when an undrifted virus circulated (2006 for H1N1 and 2008 for H3N2) but significant hospitalization was observed with a drifted or shifted virus (2004, 2005, 2007 and 2010 for H3N2, and 2009 for H1N1pdm09).ConclusionsWe documented a consistently high pediatric hospitalization burden of influenza A. Knowledge of antigenic changes and their proportion of circulation aids in the interpretation of impact of the subtypes. Year-to-year variation in hospitalization rates in young infants appeared to correlate with antigenic variation, lending support to the role of protection from maternal antibodies.

Influenza A H1N1 pandemic strain evolution--divergence and the potential for antigenic drift variants.

The emergence of a novel A(H1N1) strain in 2009 was the first influenza pandemic of the genomic age, and unprecedented surveillance of the virus provides the opportunity to better understand the evolution of influenza. We examined changes in the nucleotide coding regions and the amino acid sequences of the hemagglutinin (HA), neuraminidase (NA), and nucleoprotein (NP) segments of the A(H1N1)pdm09 strain using publicly available data. We calculated the nucleotide and amino acid hamming distance from the vaccine strain A/California/07/2009 for each sequence. We also estimated Pepitope–a measure of antigenic diversity based on changes in the epitope regions–for each isolate. Finally, we compared our results to A(H3N2) strains collected over the same period. Our analysis found that the mean hamming distance for the HA protein of the A(H1N1)pdm09 strain increased from 3.6 (standard deviation [SD]: 1.3) in 2009 to 11.7 (SD: 1.0) in 2013, while the mean hamming distance in the coding region increased from 7.4 (SD: 2.2) in 2009 to 28.3 (SD: 2.1) in 2013. These trends are broadly similar to the rate of mutation in H3N2 over the same time period. However, in contrast to H3N2 strains, the rate of mutation accumulation has slowed in recent years. Our results are notable because, over the course of the study, mutation rates in H3N2 similar to that seen with A(H1N1)pdm09 led to the emergence of two antigenic drift variants. However, while there has been an H1N1 epidemic in North America this season, evidence to date indicates the vaccine is still effective, suggesting the epidemic is not due to the emergence of an antigenic drift variant. Our results suggest that more research is needed to understand how viral mutations are related to vaccine effectiveness so that future vaccine choices and development can be more predictive.

Substitutions Near the Receptor Binding Site Determine Major Antigenic Change During Influenza Virus Evolution

The molecular basis of antigenic drift was determined for the hemagglutinin (HA) of human influenza A/H3N2 virus. From 1968 to 2003, antigenic change was caused mainly by single amino acid substitutions, which occurred at only seven positions in HA immediately adjacent to the receptor binding site. Most of these substitutions were involved in antigenic change more than once. Equivalent positions were responsible for the recent antigenic changes of influenza B and A/H1N1 viruses. Substitution of a single amino acid at one of these positions substantially changed the virus-specific antibody response in infected ferrets. These findings have potentially far-reaching consequences for understanding the evolutionary mechanisms that govern influenza viruses.

Comparative Serological Assays for the Study of H5 and H7 Avian Influenza Viruses

The nature of influenza virus to randomly mutate and evolve into new types is an important challenge in the control of influenza infection. It is necessary to monitor virus evolution for a better understanding of the pandemic risk posed by certain variants as evidenced by the highly pathogenic avian influenza (HPAI) viruses. This has been clearly recognized in Egypt following the notification of the first HPAI H5N1 outbreak. The continuous circulation of the virus and the mass vaccination programme undertaken in poultry have resulted in a progressive genetic evolution and a significant antigenic drift near the major antigenic sites. In order to establish if vaccination is sufficient to provide significant intra- and interclade cross-protection, lentiviral pseudotypes derived from H5N1 HPAI viruses (A/Vietnam/1194/04, A/chicken/Egypt-1709-01/2007) and an antigenic drift variant (A/chicken/Egypt-1709-06-2008) were constructed and used in pseudotype-based neutralization assays (pp-NT). pp-NT data obtained was confirmed and correlated with HI and MN assays. A panel of pseudotypes belonging to influenza Groups 1 and 2, with a combination of reporter systems, was also employed for testing avian sera in order to support further application of pp-NT as an alternative valid assay that can improve avian vaccination efficacy testing, vaccine virus selection, and the reliability of reference sera.

The Quadrivalent Approach to Influenza Vaccination

Antigenic distinctions between influenza A and B viruses, among various influenza A subtypes and strains, and between influenza B viruses of different lineages have posed major challenges to the development of widely effective influenza vaccines. The overall impact of disease caused by influenza B has not been fully appreciated until recently. Although less extensive than that caused by influenza A, the burden of influenza B is nonetheless substantial, and it has been responsible for both outbreaks and for severe disease, particularly in children and young adults [1–3]. Antigenic diversity is also believed to be less problematic among influenza B than among influenza A viruses. However, influenza B variants of 2 antigenically distinct lineages have been recognized since the early 1980s: prototypesB/Victoria/2/87(Victorialineage) and B/Yamagata/16/88 (Yamagata lineage). B/Yamagata was the major circulating lineage until the 1980s when B/ Victoria emerged. Since then, both influenza B virus lineages with drift variants have circulated globally, with variation as to which of the 2 influenza B lineages would be preeminent in a particular year. Historically, influenza vaccines have contained influenza B components of only 1 lineage, while having components of 2 influenzaAsubtypessince1977(A/H3N2

A “pre-seasonal” hospital outbreak of influenza pneumonia caused by the drift variant A/Victoria/361/2011-like H3N2 viruses, Hong Kong, 2011

BackgroundBeginning from late 2011 and early 2012, increasing circulation of antigenically drifted influenza A/Victoria/361/2011-like H3N2 viruses within genotype 3 of the A/Victoria/208/2009 clade have been reported in multiple European countries and elsewhere. Whether these emerging viruses are associated with increased disease severity is unclear. ObjectivesTo report the clinical and virological findings of a moderately severe hospital outbreak of A/Victoria/361/2011-like viruses that occurred in November 2011 in Hong Kong. Study designClinical and virological hospital outbreak investigation. ResultsThe outbreak occurred in an adult psychiatric ward in November 2011, a time well before the usual local seasonal influenza winter peak. Altogether, 7 patients and 1 healthcare-worker were affected (mean age, 47 [range, 34–61] years). The attack rates among patients and healthcare-workers were 33% (7/21) and 7% (1/15), respectively. Pneumonia developed in 38% (3/8) of cases; none had underlying immunocompromised conditions. High nasopharyngeal viral loads were detected. All cases responded to antiviral treatment. Multiple amino acid mutations with reference to earlier A(H3N2) vaccine strains were mapped to key antigenic sites on hemagglutinin; however, no critical mutations on receptor binding sites were detected. Viral sequence variations jeopardized the performance of molecular diagnostic assays. ConclusionsSevere disease and pneumonia occurred in a substantial proportion of non-immunocompromised adults in a hospital outbreak attributed to the emerging antigenically drifted A/Victoria/361/2011-like H3N2 viruses. Close monitoring of the transmission of this drift variant is required. Further studies are also necessary to determine virus virulence.

Molecular Characterization and Phylogenetic Analysis of the Hemagglutinin 1 Protein of Human Influenza A Virus Subtype H1N1 Circulating in Kenya During 2007-2008

Among influenza viruses, type A viruses exhibit the greatest genetic diversity, infect the widest range of host species, and cause the vast majority of cases of severe disease in humans, including cases during the great pandemics. The hemagglutinin 1 (HA1) domain of the HA protein contains the highest concentration of epitopes and, correspondingly, experiences the most intense positive selection pressure. We sought to isolate and genetically characterize influenza A virus subtype H1N1 (A[H1N1]) circulating in Kenya during 2007-2008, using the HA1 protein. Nasopharyngeal swab specimens were collected from patients aged ≥ 2 months who presented to 8 healthcare facilities in Kenya with influenza-like illness. We tested specimens for seasonal influenza A viruses, using real-time reverse-transcription polymerase chain reaction (RT-PCR). Viruses were subtyped using subtype-specific primers. Specimens positive for seasonal A(H1N1) were inoculated onto Madin-Darby canine kidney cells for virus isolation. Viral RNAs were extracted from isolates, and the HA1 gene was amplified by RT-PCR, followed by nucleotide sequencing. Nucleotide sequences were assembled using BioEdit and translated into amino acid codes, using DS Gene, version 1.5. Multiple sequence alignments were performed using MUSCLE, version 3.6, and phylogenetic analysis was performed using MrBayes software. We found that, similar to A/Brisbane/59/2007 (H1N1)-like virus, which was included in the southern hemisphere vaccine for the 2009 influenza season, all 2007 Kenyan viruses had D39N, R77K, T132V, K149R, and E277K amino acid substitutions, compared with A/Solomon Islands/3/2006 (H1N1)-like virus, a component of the southern hemisphere vaccine for the 2008 influenza season. However, the majority of 2008 viruses from Kenya also had R192K and R226Q substitutions, compared with A/Solomon Islands/3/2006 (H1N1)-like virus. These 2 changes occurred at the receptor binding site. The majority of the 2008 Kenyan isolates contained N187S, G189N, and A193T mutations, which differed from A/Brisbane/59/2007 (H1N1)-like virus. The A193T substitution is involved in binding the sialic acid receptor. Phylogenetically, the 2008 Kenyan isolates grouped into 2 clusters. The main cluster contained viruses with N187S and A193T changes; residue 187 is involved in receptor binding, whereas residue 193 is at antigenic site Sb. Overall, the major genetic variations that occurred in seasonal A(H1) viruses either affected receptor binding or altered epitopes at the immunodominant sites. These genetic variations in seasonal A(H1N1) isolated in Kenya during 2007-2008 highlight the importance of continuing surveillance and characterization of emerging drift variants of influenza virus in Africa.

Comparison of the Cross-Reactive Anti-Influenza Neutralizing Activity of Polymeric and Monomeric IgA Monoclonal Antibodies

Here we examined whether polymeric IgA (pIgA) and monomeric IgA (mIgA) antibodies differ in their ability to neutralize drift viruses within the same subtype. We used an IgA monoclonal antibody (mAb; H1-21) against influenza virus strain A/Hiroshima/52/2005 (A/Hiroshima; H3N2). The mAb was obtained after immunizing mice mucosally with a split-virion (SV) vaccine. The mAb contained both mIgA and pIgA forms. It reacted with the homologous virus and cross-reacted with drift viruses A/New York/55/2004 (H3N2) and A/Wyoming/3/2003 (H3N2) in hemagglutinin-inhibition (HI) and neutralizing Ab assays. The mAb also cross-reacted with A/Panama/2007/99 (H3N2) in an ELISA. We separated the mAb into pIgA and mIgA fractions by gel filtration, and then tested them for neutralizing Ab activity. The neutralizing activity for the A/Hiroshima/52/2005, A/New York/55/2004, and A/Wyoming/3/2003 viruses was lower for the mIgA than the pIgA fraction. However, the neutralizing efficiency for drift variants relative to that for the homotype did not differ between pIgA and mIgA, and pIgA only neutralized variants that could also be neutralized by mIgA. These results suggest that the polymerization of IgA enhances its antiviral immune responses, but does not increase the number of influenza virus strains neutralized by the IgA.