Longitudinal sampling is required to maximize detection of intrahost A/H3N2 virus variants

Abstract Seasonal human influenza viruses continually change antigenically to escape from neutralizing antibodies. It remains unclear how genetic variation in the intrahost virus population and selection at the level of individual hosts translates to the fast-paced evolution observed at the global level because emerging intrahost antigenic variants are rarely detected. We tracked intrahost variants in the hemagglutinin and neuraminidase surface proteins using longitudinally collected samples from 52 patients infected by A/H3N2 influenza virus, mostly young children, who received oseltamivir treatment. We identified emerging putative antigenic variants and oseltamivir-resistant variants, most of which remained detectable in samples collected at subsequent days, and identified variants that emerged intrahost immediately prior to increases in global rates. In contrast to most putative antigenic variants, oseltamivir-resistant variants rapidly increased to high frequencies in the virus population. Importantly, the majority of putative antigenic variants and oseltamivir-resistant variants were first detectable four or more days after onset of symptoms or start of treatment, respectively. Our observations demonstrate that de novo variants emerge, and may be positively selected, during the course of infection. Additionally, based on the 4–7 days post-treatment delay in emergence of oseltamivir-resistant variants in six out of the eight individuals with such variants, we find that limiting sample collection for routine surveillance and diagnostic testing to early timepoints after onset of symptoms can potentially preclude detection of emerging, positively selected variants.

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Middle-aged individuals may be in a perpetual state of H3N2 influenza virus susceptibility

10.1101/2020.01.09.20017038 ◽

2020 ◽

Cited By ~ 1

Author(s):

Sigrid Gouma ◽

Kangchon Kim ◽

Madison Weirick ◽

Megan E. Gumina ◽

Angela Branche ◽

...

Keyword(s):

Neutralizing Antibodies ◽

Influenza Viruses ◽

H3n2 Virus ◽

Virus Infections ◽

Middle Aged ◽

Viral Susceptibility ◽

Antibody Titers ◽

1960S And 1970S ◽

H3n2 Influenza ◽

The 1960S

Most humans are infected with influenza viruses by 3-4 years of age (1) and have high antibody titers against viral strains encountered early in life (2). Early childhood influenza exposures can leave lifelong ‘immunological imprints’ that affect how an individual responds to antigenically distinct viral strains later in life (3,4). H3N2 influenza viruses began circulating in humans in 1968 and have evolved substantially over the past 51 years (5). Therefore, an individual’s birth year largely predicts which specific type of H3N2 virus they first encountered in childhood. Here, we completed a large serological survey to elucidate the specificity of antibodies against contemporary H3N2 viruses in differently aged individuals who were likely primed with different H3N2 strains in childhood. We found that most humans who were first infected in childhood with H3N2 viral strains from the 1960s and 1970s possess non-neutralizing antibodies against contemporary 3c2.A H3N2 viruses. Most importantly, we found that 3c2.A H3N2 virus infections boost non-neutralizing H3N2 antibodies in middle-aged individuals, potentially leaving many of them in a perpetual state of 3c2.A H3N2 viral susceptibility.

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Influenza Virus Resistance to Human Neutralizing Antibodies

mBio ◽

10.1128/mbio.00213-12 ◽

2012 ◽

Vol 3 (4) ◽

Author(s):

James E. Crowe

Keyword(s):

Influenza Virus ◽

B Cell ◽

Neutralizing Antibodies ◽

Cell Receptor ◽

B Cell Receptor ◽

Influenza Viruses ◽

Surface Proteins ◽

Progeny Virus ◽

Human Antibody ◽

Functional Surface

ABSTRACT The human antibody repertoire has an exceptionally large capacity to recognize new or changing antigens through combinatorial and junctional diversity established at the time of V(D)J recombination and through somatic hypermutation. Influenza viruses exhibit a relentless capacity to escape the human antibody response by altering the amino acids of their surface proteins in hypervariable domains that exhibit a high level of structural plasticity. Both parties in this high-stakes game of shape shifting drive structural evolution of their functional proteins (the B cell receptor/antibody on one side and the viral hemagglutinin and neuraminidase proteins on the other) using error-prone polymerase systems. It is likely that most of the genetic mutations that occur in these systems are deleterious, resulting in the failure of the B cell or virus with mutations to propagate in the immune repertoire or viral quasispecies. A subset of mutations is tolerated in functional surface proteins that enter the B cell or virus progeny pool. In both cases, selection occurs in the population of mutated and unmutated species. In cases where the functional avidity of the B cell receptor is increased significantly, that clone may be selected for preferential expansion. In contrast, an influenza virus that “escapes” the inhibitory effect of secreted antibodies may represent a high proportion of the progeny virus in that host. The recent paper by O’Donnell et al. [C. D. O’Donnell et al., mBio 3(3):e00120-12, 2012] identifies a mechanism for antibody resistance that does not require escape from binding but rather achieves a greater efficiency in replication.

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Comparison of A(H3N2) Neutralizing Antibody Responses Elicited by 2018–2019 Season Quadrivalent Influenza Vaccines Derived from Eggs, Cells, and Recombinant Hemagglutinin

Clinical Infectious Diseases ◽

10.1093/cid/ciaa1352 ◽

2020 ◽

Author(s):

Wei Wang ◽

Esmeralda Alvarado-Facundo ◽

Russell Vassell ◽

Limone Collins ◽

Rhonda E Colombo ◽

...

Keyword(s):

Neutralizing Antibodies ◽

Neutralizing Antibody ◽

Vaccine Virus ◽

Influenza Viruses ◽

Antigenic Drift ◽

Influenza Vaccines ◽

Open Label ◽

Virus Propagation ◽

Vaccine Antigens ◽

H3n2 Influenza

Abstract Background Low vaccine effectiveness against A(H3N2) influenza in seasons with little antigenic drift has been attributed to substitutions in hemagglutinin (HA) acquired during vaccine virus propagation in eggs. Clinical trials comparing recombinant HA vaccine (rHA) and cell-derived inactivated influenza vaccine (IIV) to egg-derived IIVs provide opportunities to assess how egg-adaptive substitutions influence HA immunogenicity. Methods Neutralization titers in pre- and postimmunization sera from 133 adults immunized with 1 of 3 types of influenza vaccines in a randomized, open-label trial during the 2018–2019 influenza season were measured against egg- and cell-derived A/Singapore/INFIMH-16-0019/2016-like and circulating A(H3N2) influenza viruses using HA pseudoviruses. Results All vaccines elicited neutralizing antibodies to all H3 vaccine antigens, but the rHA vaccine elicited the highest titers and seroconversion rates against all strains tested. Egg- and cell-derived IIVs elicited responses similar to each other. Preimmunization titers against H3 HA pseudoviruses containing egg-adaptive substitutions T160K and L194P were high, but lower against H3 HA pseudoviruses without those substitutions. All vaccines boosted neutralization titers against HA pseudoviruses with egg-adaptive substitutions, but poorly neutralized wild-type 2019–2020 A/Kansas/14/2017 (H3N2) HA pseudoviruses. Conclusion Egg- and cell-derived 2018–2019 season influenza vaccines elicited similar neutralization titers and response rates, indicating that the cell-derived vaccine did not improve immunogenicity against the A(H3N2) viruses. The higher responses after rHA vaccination may be due to its higher HA content. All vaccines boosted titers to HA with egg-adaptive substitutions, suggesting boosting from past antigens or better exposure of HA epitopes. Studies comparing immunogenicity and effectiveness of different influenza vaccines across many seasons are needed.

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

PLoS Pathogens ◽

10.1371/journal.ppat.1009273 ◽

2021 ◽

Vol 17 (2) ◽

pp. e1009273

Author(s):

Valerie Le Sage ◽

Jennifer E. Jones ◽

Karen A. Kormuth ◽

William J. Fitzsimmons ◽

Eric Nturibi ◽

...

Keyword(s):

Neutralizing Antibodies ◽

Natural Infection ◽

Adaptive Immune Response ◽

Influenza Viruses ◽

H3n2 Virus ◽

Influenza B Virus ◽

Influenza B ◽

Respiratory Pathogens ◽

Airborne Transmission ◽

Heterosubtypic Immunity

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.

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Contemporary H3N2 influenza viruses have a glycosylation site that alters binding of antibodies elicited by egg-adapted vaccine strains

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1712377114 ◽

2017 ◽

Vol 114 (47) ◽

pp. 12578-12583 ◽

Cited By ~ 231

Author(s):

Seth J. Zost ◽

Kaela Parkhouse ◽

Megan E. Gumina ◽

Kangchon Kim ◽

Sebastian Diaz Perez ◽

...

Keyword(s):

Seasonal Influenza ◽

Influenza Season ◽

Antigenic Site ◽

Glycosylation Site ◽

Influenza Viruses ◽

Antibody Responses ◽

H3n2 Virus ◽

Viral Strain ◽

H3n2 Influenza ◽

H3n2 Vaccine

H3N2 viruses continuously acquire mutations in the hemagglutinin (HA) glycoprotein that abrogate binding of human antibodies. During the 2014–2015 influenza season, clade 3C.2a H3N2 viruses possessing a new predicted glycosylation site in antigenic site B of HA emerged, and these viruses remain prevalent today. The 2016–2017 seasonal influenza vaccine was updated to include a clade 3C.2a H3N2 strain; however, the egg-adapted version of this viral strain lacks the new putative glycosylation site. Here, we biochemically demonstrate that the HA antigenic site B of circulating clade 3C.2a viruses is glycosylated. We show that antibodies elicited in ferrets and humans exposed to the egg-adapted 2016–2017 H3N2 vaccine strain poorly neutralize a glycosylated clade 3C.2a H3N2 virus. Importantly, antibodies elicited in ferrets infected with the current circulating H3N2 viral strain (that possesses the glycosylation site) and humans vaccinated with baculovirus-expressed H3 antigens (that possess the glycosylation site motif) were able to efficiently recognize a glycosylated clade 3C.2a H3N2 virus. We propose that differences in glycosylation between H3N2 egg-adapted vaccines and circulating strains likely contributed to reduced vaccine effectiveness during the 2016–2017 influenza season. Furthermore, our data suggest that influenza virus antigens prepared via systems not reliant on egg adaptations are more likely to elicit protective antibody responses that are not affected by glycosylation of antigenic site B of H3N2 HA.

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Immunodominance of Antigenic Site B in the Hemagglutinin of the Current H3N2 Influenza Virus in Humans and Mice

Journal of Virology ◽

10.1128/jvi.01100-18 ◽

2018 ◽

Vol 92 (20) ◽

Cited By ~ 13

Author(s):

Felix Broecker ◽

Sean T. H. Liu ◽

Weina Sun ◽

Florian Krammer ◽

Viviana Simon ◽

...

Keyword(s):

Influenza Virus ◽

Hemagglutination Inhibition ◽

Neutralizing Antibodies ◽

Antigenic Site ◽

Influenza Viruses ◽

Antigenic Sites ◽

Head Domain ◽

Hemagglutinin Protein ◽

H3n2 Influenza ◽

H3n2 Influenza Virus

ABSTRACTThe hemagglutinin protein of H3N2 influenza viruses is the major target of neutralizing antibodies induced by infection and vaccination. However, the virus frequently escapes antibody-mediated neutralization due to mutations in the globular head domain. Five topologically distinct antigenic sites in the head domain of H3 hemagglutinin, A to E, have been previously described by mapping the binding sites of monoclonal antibodies, yet little is known about the contribution of each site to the immunogenicity of modern H3 hemagglutinins, as measured by hemagglutination inhibition activity, which is known to correlate with protection. To investigate the hierarchy of antibody immunodominance, five Δ1 recombinant influenza viruses expressing hemagglutinin of the A/Hong Kong/4801/2014 (H3N2) strain with mutations in single antigenic sites were generated. Next, the Δ1 viruses were used to determine the hierarchy of immunodominance by measuring the hemagglutination inhibition reactivity of mouse antisera and plasma from 18 human subjects before and after seasonal influenza vaccination in 2017-2018. In both mice and humans, mutations in antigenic site B caused the most significant decrease in hemagglutination inhibition titers compared to wild-type hemagglutinin. This study revealed that antigenic site B is immunodominant in the H3N2 influenza virus strain included in the current vaccine preparations.IMPORTANCEInfluenza viruses rapidly evade humoral immunity through antigenic drift, making current vaccines poorly effective and antibody-mediated protection short-lived. The majority of neutralizing antibodies target five antigenic sites in the head domain of the hemagglutinin protein that are also the most sequence-variable regions. A better understanding of the contribution of each antigenic site to the overall antibody response to hemagglutinin may help in the design of improved influenza virus vaccines.

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Development of Universal Influenza Vaccines Targeting Conserved Viral Proteins

Vaccines ◽

10.3390/vaccines7040169 ◽

2019 ◽

Vol 7 (4) ◽

pp. 169 ◽

Cited By ~ 8

Author(s):

Jazayeri ◽

Poh

Keyword(s):

Immune Responses ◽

Influenza Vaccine ◽

Neutralizing Antibodies ◽

Influenza Viruses ◽

Surface Proteins ◽

Influenza Vaccines ◽

Influenza Pandemics ◽

Host Immune Responses ◽

Production Platform ◽

Universal Influenza Vaccine

Vaccination is still the most efficient way to prevent an infection with influenza viruses. Nevertheless, existing commercial vaccines face serious limitations such as availability during epidemic outbreaks and their efficacy. Existing seasonal influenza vaccines mostly induce antibody responses to the surface proteins of influenza viruses, which frequently change due to antigenic shift and or drift, thus allowing influenza viruses to avoid neutralizing antibodies. Hence, influenza vaccines need a yearly formulation to protect against new seasonal viruses. A broadly protective or universal influenza vaccine must induce effective humoral as well as cellular immunity against conserved influenza antigens, offer good protection against influenza pandemics, be safe, and have a fast production platform. Nanotechnology has great potential to improve vaccine delivery, immunogenicity, and host immune responses. As new strains of human epidemic influenza virus strains could originate from poultry and swine viruses, development of a new universal influenza vaccine will require the immune responses to be directed against viruses from different hosts. This review discusses how the new vaccine platforms and nanoparticles can be beneficial in the development of a broadly protective, universal influenza vaccine.

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Influenza classification from short reads with VAPOR facilitates robust mapping pipelines and zoonotic strain detection for routine surveillance applications

Bioinformatics ◽

10.1093/bioinformatics/btz814 ◽

2019 ◽

Vol 36 (6) ◽

pp. 1681-1688

Author(s):

Joel A Southgate ◽

Matthew J Bull ◽

Clare M Brown ◽

Joanne Watkins ◽

Sally Corden ◽

...

Keyword(s):

De Novo ◽

Influenza Viruses ◽

Supplementary Information ◽

Global Public Health ◽

Human Origin ◽

Short Read ◽

Public Health Burden ◽

Routine Surveillance ◽

Surveillance Applications ◽

Strain Detection

Abstract Motivation Influenza viruses represent a global public health burden due to annual epidemics and pandemic potential. Due to a rapidly evolving RNA genome, inter-species transmission, intra-host variation, and noise in short-read data, reads can be lost during mapping, and de novo assembly can be time consuming and result in misassembly. We assessed read loss during mapping and designed a graph-based classifier, VAPOR, for selecting mapping references, assembly validation and detection of strains of non-human origin. Results Standard human reference viruses were insufficient for mapping diverse influenza samples in simulation. VAPOR retrieved references for 257 real whole-genome sequencing samples with a mean of >99.8% identity to assemblies, and increased the proportion of mapped reads by up to 13.3% compared to standard references. VAPOR has the potential to improve the robustness of bioinformatics pipelines for surveillance and could be adapted to other RNA viruses. Availability and implementation VAPOR is available at https://github.com/connor-lab/vapor. Supplementary information Supplementary data are available at Bioinformatics online.

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Synergistic PA and HA mutations confer mouse adaptation of a contemporary A/H3N2 influenza virus

Scientific Reports ◽

10.1038/s41598-019-51877-4 ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 2

Author(s):

Mariana Baz ◽

Zeineb M’hamdi ◽

Julie Carbonneau ◽

Sophie Lavigne ◽

Christian Couture ◽

...

Keyword(s):

Animal Model ◽

Influenza Virus ◽

Reverse Genetics ◽

Polymerase Activity ◽

Influenza Viruses ◽

H3n2 Virus ◽

Wild Type Virus ◽

Virus Research ◽

H3n2 Influenza

Abstract The mouse is the most widely used animal model for influenza virus research. However, the susceptibility of mice to seasonal influenza virus depends on the strain of mouse and on the strain of the influenza virus. Seasonal A/H3N2 influenza viruses do not replicate well in mice and therefore they need to be adapted to this animal model. In this study, we generated a mouse-adapted A/H3N2 virus (A/Switzerland/9715293/2013 [MA-H3N2]) by serial passaging in mouse lungs that exhibited greater virulence compared to the wild-type virus (P0-H3N2). Seven mutations were found in the genome of MA-H3N2: PA(K615E), NP(G384R), NA(G320E) and HA(N122D, N144E, N246K, and A304T). Using reverse genetics, two synergistically acting genes were found as determinants of the pathogenicity in mice. First, the HA substitutions were shown to enhanced viral replication in vitro and, second, the PA-K615E substitution increased polymerase activity, although did not alter virus replication in vitro or in mice. Notably, single mutations had only limited effects on virulence in vitro. In conclusion, a co-contribution of HA and PA mutations resulted in a lethal mouse model of seasonal A/H3N2 virus. Such adapted virus is an excellent tool for evaluation of novel drugs or vaccines and for study of influenza pathogenesis.

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Pathogenicity and Transmissibility of Novel Reassortant H3N2 Influenza Viruses with 2009 Pandemic H1N1 Genes in Pigs

Journal of Virology ◽

10.1128/jvi.03355-14 ◽

2014 ◽

Vol 89 (5) ◽

pp. 2831-2841 ◽

Cited By ~ 24

Author(s):

Jingjiao Ma ◽

Huigang Shen ◽

Qinfang Liu ◽

Bhupinder Bawa ◽

Wenbao Qi ◽

...

Keyword(s):

Binding Site ◽

Receptor Binding ◽

The United States ◽

Influenza Viruses ◽

H3n2 Virus ◽

Pandemic H1n1 ◽

M Gene ◽

Receptor Binding Site ◽

H3n2 Influenza ◽

Swine Herds

ABSTRACTAt least 10 different genotypes of novel reassortant H3N2 influenza viruses with 2009 pandemic H1N1 [A(H1N1)pdm09] gene(s) have been identified in U.S. pigs, including the H3N2 variant with a single A(H1N1)pdm09 M gene, which has infected more than 300 people. To date, only three genotypes of these viruses have been evaluated in animal models, and the pathogenicity and transmissibility of the other seven genotype viruses remain unknown. Here, we show that three H3N2 reassortant viruses that contain 3 (NP, M, and NS) or 5 (PA, PB2, NP, M, and NS) genes from A(H1N1)pdm09 were pathogenic in pigs, similar to the endemic H3N2 swine virus. However, the reassortant H3N2 virus with 3 A(H1N1)pdm09 genes and a recent human influenza virus N2 gene was transmitted most efficiently among pigs, whereas the reassortant H3N2 virus with 5 A(H1N1)pdm09 genes was transmitted less efficiently than the endemic H3N2 virus. Interestingly, the polymerase complex of reassortant H3N2 virus with 5 A(H1N1)pdm09 genes showed significantly higher polymerase activity than those of endemic and reassortant H3N2 viruses with 3 A(H1N1)pdm09 genes. Further studies showed that an avian-like glycine at position 228 at the hemagglutinin (HA) receptor binding site is responsible for inefficient transmission of the reassortant H3N2 virus with 5 A(H1N1)pdm09 genes. Taken together, our results provide insights into the pathogenicity and transmissibility of novel reassortant H3N2 viruses in pigs and suggest that a mammalian-like serine at position 228 in the HA is critical for the transmissibility of these reassortant H3N2 viruses.IMPORTANCESwine influenza is a highly contagious zoonotic disease that threatens animal and public health. Introduction of 2009 pandemic H1N1 virus [A(H1N1)pdm09] into swine herds has resulted in novel reassortant influenza viruses in swine, including H3N2 and H1N2 variants that have caused human infections in the United States. We showed that reassortant H3N2 influenza viruses with 3 or 5 genes from A(H1N1)pdm09 isolated from diseased pigs are pathogenic and transmissible in pigs, but the reassortant H3N2 virus with 5 A(H1N1)pdm09 genes displayed less efficient transmissibility than the endemic and reassortant H3N2 viruses with 3 A(H1N1)pdm09 genes. Further studies revealed that an avian-like glycine at the HA 228 receptor binding site of the reassortant H3N2 virus with 5 A(H1N1)pdm09 genes is responsible for less efficient transmissibility in pigs. Our results provide insights into viral pathogenesis and the transmission of novel reassortant H3N2 viruses that are circulating in U.S. swine herds and warrant future surveillance.

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