Unmasking Stem-Specific Neutralizing Epitopes by Abolishing N-Linked Glycosylation Sites of Influenza Virus Hemagglutinin Proteins for Vaccine Design

ABSTRACTInfluenza virus hemagglutinin (HA) protein consists of two components, i.e., a globular head region and a stem region that are folded within six disulfide bonds, plus several N-linked glycans that produce a homotrimeric complex structure. While N-linked glycosylation sites on the globular head are variable among different strains and different subtypes, N-linked glycosylation sites in the stem region are mostly well conserved among various influenza virus strains. Targeting highly conserved HA stem regions has been proposed as a useful strategy for designing universal influenza vaccines. Since the HA stem region is constituted by an HA1 N-terminal part and a full HA2 part, we expressed a series of recombinant HA mutant proteins with deleted N-linked glycosylation sites in the HA1 stem and HA2 stem regions of H5N1 and pH1N1 viruses. Unmasking N-glycans in the HA2 stem region (H5 N484A and H1 N503A) was found to elicit more potent neutralizing antibody titers against homologous, heterologous, and heterosubtypic viruses. Unmasking the HA2 stem N-glycans of H5HA but not H1HA resulted in more CR6261-like and FI6v3-like antibodies and also correlated with the increase of cell fusion inhibition activity in antisera. Only H5 N484A HA2 stem mutant protein immunization increased the numbers of antibody-secreting cells, germinal center B cells, and memory B cells targeting the stem helix A epitopes in splenocytes. Unmasking the HA2 stem N-glycans of H5HA mutant proteins showed a significantly improvement in the protection against homologous virus challenges but did so to a less degree for the protection against heterosubtypic pH1N1 virus challenges. These results may provide useful information for designing more effective influenza vaccines.IMPORTANCEN-linked glycosylation sites in the stem regions of influenza virus hemagglutinin (HA) proteins are mostly well conserved among various influenza virus strains. Targeting highly conserved HA stem regions has been proposed as a useful strategy for designing universal influenza vaccines. Our studies indicate that unmasking the HA2 stem N-glycans of recombinant HA proteins from H5N1 and pH1N1 viruses induced more potent neutralizing antibody titers against homologous and heterosubtypic viruses. However, only immunization with the H5N1 HA2 stem mutant protein can refocus B antibody responses to the helix A epitope for inducing more CR6261-like/FI6v3-like and fusion inhibition antibodies in antisera, resulting in a significant improvement for the protection against lethal H5N1 virus challenges. These results may provide useful information for designing more effective influenza vaccines.

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Neuraminidase-Inhibiting Antibody Titers Correlate with Protection from Heterologous Influenza Virus Strains of the Same Neuraminidase Subtype

Journal of Virology ◽

10.1128/jvi.01006-18 ◽

2018 ◽

Vol 92 (17) ◽

Cited By ~ 10

Author(s):

Lisa Walz ◽

Sarah-Katharina Kays ◽

Gert Zimmer ◽

Veronika von Messling

Keyword(s):

Influenza Virus ◽

Immune Responses ◽

Vesicular Stomatitis Virus ◽

Influenza Viruses ◽

Antigenic Drift ◽

Influenza Vaccines ◽

Vaccine Antigen ◽

Antibody Titers ◽

Vesicular Stomatitis ◽

Virus Strains

ABSTRACTImmune responses induced by currently licensed inactivated influenza vaccines are mainly directed against the hemagglutinin (HA) glycoprotein, the immunodominant antigen of influenza viruses. The resulting antigenic drift of HA requires frequent updating of the vaccine composition and annual revaccination. On the other hand, the levels of antibodies directed against the neuraminidase (NA) glycoprotein, the second major influenza virus antigen, vary greatly. To investigate the potential of the more conserved NA protein for the induction of subtype-specific protection, vesicular stomatitis virus-based replicons expressing a panel of N1 proteins from prototypic seasonal and pandemic H1N1 strains and human H5N1 and H7N9 isolates were generated. Immunization of mice and ferrets with the replicon carrying the matched N1 protein resulted in robust humoral and cellular immune responses and protected against challenge with the homologous influenza virus with an efficacy similar to that of the matched HA protein, illustrating the potential of the NA protein as a vaccine antigen. The extent of protection after immunization with mismatched N1 proteins correlated with the level of cross-reactive neuraminidase-inhibiting antibody titers. Passive serum transfer experiments in mice confirmed that these functional antibodies determine subtype-specific cross-protection. Our findings illustrate the potential of NA-specific immunity for achieving broader protection against antigenic drift variants or newly emerging viruses carrying the same NA but a different HA subtype.IMPORTANCEDespite the availability of vaccines, annual influenza virus epidemics cause 250,000 to 500,000 deaths worldwide. Currently licensed inactivated vaccines, which are standardized for the amount of the hemagglutinin (HA) antigen, primarily induce strain-specific antibodies, whereas the immune response to the neuraminidase (NA) antigen, which is also present on the viral surface, is usually low. Using NA-expressing single-cycle vesicular stomatitis virus replicons, we show that the NA antigen conferred protection of mice and ferrets against not only the matched influenza virus strains but also viruses carrying NA proteins from other strains of the same subtype. The extent of protection correlated with the level of cross-reactive NA-inhibiting antibodies. This highlights the potential of the NA antigen for the development of more broadly protective influenza vaccines. Such vaccines may also provide partial protection against newly emerging strains with the same NA but a different HA subtype.

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The antibody landscapes against Group 1 and 2 influenza virus hemagglutinin following AS03 and MF59 adjuvanted H5N1 vaccination

10.1101/2021.07.09.21259780 ◽

2021 ◽

Author(s):

Johannes B Goll ◽

Aarti Jain ◽

Travis L Jensen ◽

Rafael Assis ◽

Rie Nakajima ◽

...

Keyword(s):

Influenza Virus ◽

Pandemic Influenza ◽

Influenza A ◽

Antibody Responses ◽

Influenza Vaccines ◽

Influenza B ◽

Vaccine Adjuvants ◽

Influenza Virus Hemagglutinin ◽

Globular Head ◽

Group 1

Current seasonal and pre-pandemic influenza vaccines induce short-lived predominantly strain-specific and limited heterosubtypic responses. To better understand how vaccine adjuvants AS03 and MF59 may provide improved antibody responses to vaccination, we interrogated serum from subjects who received 2 doses of inactivated monovalent influenza A/Indonesia/05/2005 vaccine with or without AS03 or MF59 using hemagglutinin (HA) microarrays. The arrays were designed to reflect both full length and globular head HA proteins derived from 17 influenza A subtypes (H1 to H16 and H18) and influenza B strains. We observed significantly increased strain-specific and broad homo- and hetero-subtypic antibody responses with both AS03 and MF59 adjuvanted vaccination with AS03 achieving a higher titer and breadth of IgG responses relative to MF59. Adjuvanted vaccine was also associated with the elicitation of stalk directed antibody. Finally, we established good correlation of the array antibody responses to H5 antigens with standard hemagglutination inhibition and microneutralization titers.

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Influenza Virus Vaccine Based on the Conserved Hemagglutinin Stalk Domain

mBio ◽

10.1128/mbio.00018-10 ◽

2010 ◽

Vol 1 (1) ◽

Cited By ~ 348

Author(s):

John Steel ◽

Anice C. Lowen ◽

Taia T. Wang ◽

Mark Yondola ◽

Qinshan Gao ◽

...

Keyword(s):

Influenza Virus ◽

Virus Vaccine ◽

Neutralizing Antibodies ◽

Influenza Virus Vaccine ◽

Influenza Vaccines ◽

Host Immune System ◽

Stalk Domain ◽

Further Development ◽

Virus Strains ◽

Globular Head

ABSTRACTAlthough highly effective in the general population when well matched to circulating influenza virus strains, current influenza vaccines are limited in their utility due to the narrow breadth of protection they provide. The strain specificity of vaccines presently in use mirrors the exquisite specificity of the neutralizing antibodies that they induce, that is, antibodies which bind to the highly variable globular head domain of hemagglutinin (HA). Herein, we describe the construction of a novel immunogen comprising the conserved influenza HA stalk domain and lacking the globular head. Vaccination of mice with this headless HA construct elicited immune sera with broader reactivity than those obtained from mice immunized with a full-length HA. Furthermore, the headless HA vaccine provided full protection against death and partial protection against disease following lethal viral challenge. Our results suggest that the response induced by headless HA vaccines is sufficiently potent to warrant their further development toward a universal influenza virus vaccine.IMPORTANCECurrent influenza vaccines are effective against only a narrow range of influenza virus strains. It is for this reason that new vaccines must be generated and administered each year. We now report progress toward the goal of an influenza virus vaccine which would protect against multiple strains. Our approach is based on presentation to the host immune system of a region of the influenza virus—called a “headless hemagglutinin” (headless HA)—which is similar among a multitude of diverse strains. We show that vaccination of mice with a headless HA confers protection to these animals against a lethal influenza virus challenge, thereby demonstrating the viability of the approach. Through further development and testing, we predict that a single immunization with a headless HA vaccine will offer effective protection through several influenza epidemics.

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Inhibition of ciliary activity in organ cultures of ferret trachea in reference to genetic and biological characters of influenza virus strains

Canadian Journal of Microbiology ◽

10.1139/m82-122 ◽

1982 ◽

Vol 28 (7) ◽

pp. 809-814 ◽

Cited By ~ 3

Author(s):

P. Diaz-Rodriguez ◽

A. Boudreault

Keyword(s):

Influenza Virus ◽

Influenza Viruses ◽

Influenza Vaccines ◽

Ciliary Activity ◽

Organ Cultures ◽

Preliminary Screening ◽

Cold Adapted ◽

Activity Inhibition ◽

Biological Characters ◽

Virus Strains

As reported previously, attenuated stable inhibitor-resistant influenza viruses can be screened by a 50% ciliary activity inhibition test in ferret tracheal organ cultures. This test was further applied to 5 attenuated cold-adapted influenza strains and to 11 strains with known a percentage of RNA–RNA hybridization with the parental A/PR/8/34 (H0N1) virus strain. Again, with one exception, attenuated strains could be clearly differentiated from virulent ones. It was concluded that virulence of influenza strains for man can be detected using this test regardless of the techniques used to prepare attenuated variants. A preliminary screening of attenuated candidates for live influenza vaccines can be achieved with confidence on ferret tracheal organ cultures.

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A public broadly neutralizing antibody class targets a membrane-proximal anchor epitope of influenza virus hemagglutinin

10.1101/2021.02.25.432905 ◽

2021 ◽

Author(s):

Jenna J. Guthmiller ◽

Julianna Han ◽

Henry A. Utset ◽

Lei Li ◽

Linda Yu-Ling Lan ◽

...

Keyword(s):

Influenza Virus ◽

Virus Vaccine ◽

Neutralizing Antibodies ◽

Neutralizing Antibody ◽

Human Memory ◽

Influenza Virus Vaccine ◽

Virus Infections ◽

Broadly Neutralizing Antibodies ◽

Cell Repertoire ◽

Influenza Virus Hemagglutinin

SummaryBroadly neutralizing antibodies against influenza virus hemagglutinin (HA) have the potential to provide universal protection against influenza virus infections. Here, we report a distinct class of broadly neutralizing antibodies targeting an epitope toward the bottom of the HA stalk domain where HA is “anchored” to the viral membrane. Antibodies targeting this membrane-proximal anchor epitope utilized a highly restricted repertoire, which encode for two conserved motifs responsible for HA binding. Anchor targeting B cells were common in the human memory B cell repertoire across subjects, indicating pre-existing immunity against this epitope. Antibodies against the anchor epitope at both the serological and monoclonal antibody levels were potently induced in humans by a chimeric HA vaccine, a potential universal influenza virus vaccine. Altogether, this study reveals an underappreciated class of broadly neutralizing antibodies against H1-expressing viruses that can be robustly recalled by a candidate universal influenza virus vaccine.

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Response of Influenza Vaccines Against Heterovariant Influenza Virus Strains in Adults with Chronic Diseases

Journal of Clinical Immunology ◽

10.1007/s10875-007-9100-4 ◽

2007 ◽

Vol 27 (5) ◽

pp. 542-547 ◽

Cited By ~ 30

Author(s):

V. Baldo ◽

T. Baldovin ◽

A. Floreani ◽

E. Fragapane ◽

R. Trivello ◽

...

Keyword(s):

Influenza Virus ◽

Chronic Diseases ◽

Influenza Vaccines ◽

Virus Strains

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A Prevalent Focused Human Antibody Response to the Influenza Virus Hemagglutinin Head Interface

mBio ◽

10.1128/mbio.01144-21 ◽

2021 ◽

Author(s):

Kevin R. McCarthy ◽

Jiwon Lee ◽

Akiko Watanabe ◽

Masayuki Kuraoka ◽

Lindsey R. Robinson-McCarthy ◽

...

Keyword(s):

Influenza Virus ◽

Herd Immunity ◽

Influenza Viruses ◽

Influenza Vaccines ◽

Human Antibody ◽

Human Influenza ◽

Human Antibodies ◽

Influenza Virus Hemagglutinin ◽

Selection For ◽

Rapid Appearance

The rapid appearance of mutations in circulating human influenza viruses and selection for escape from herd immunity require prediction of likely variants for an annual updating of influenza vaccines. The identification of human antibodies that recognize conserved surfaces on the influenza virus hemagglutinin (HA) has prompted efforts to design immunogens that might selectively elicit such antibodies.

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Assessment of population susceptibility to upcoming seasonal influenza epidemic strain using interepidemic emerging influenza virus strains

Epidemiology and Infection ◽

10.1017/s0950268819001717 ◽

2019 ◽

Vol 147 ◽

Cited By ~ 5

Author(s):

Lin-Lei Chen ◽

Wai-Lan Wu ◽

Wan-Mui Chan ◽

Carol H. Y. Fong ◽

Anthony C. K. Ng ◽

...

Keyword(s):

Influenza Virus ◽

Seasonal Influenza ◽

Influenza Season ◽

Geometric Mean ◽

Influenza Epidemic ◽

Multifaceted Approach ◽

Fold Reduction ◽

Significant Difference ◽

Antibody Titers ◽

Virus Strains

Abstract Seasonal influenza virus epidemics have a major impact on healthcare systems. Data on population susceptibility to emerging influenza virus strains during the interepidemic period can guide planning for resource allocation of an upcoming influenza season. This study sought to assess the population susceptibility to representative emerging influenza virus strains collected during the interepidemic period. The microneutralisation antibody titers (MN titers) of a human serum panel against representative emerging influenza strains collected during the interepidemic period before the 2018/2019 winter influenza season (H1N1-inter and H3N2-inter) were compared with those against influenza strains representative of previous epidemics (H1N1-pre and H3N2-pre). A multifaceted approach, incorporating both genetic and antigenic data, was used in selecting these representative influenza virus strains for the MN assay. A significantly higher proportion of individuals had a ⩾four-fold reduction in MN titers between H1N1-inter and H1N1-pre than that between H3N2-inter and H3N2-pre (28.5% (127/445) vs. 4.9% (22/445), P < 0.001). The geometric mean titer (GMT) of H1N1-inter was significantly lower than that of H1N1-pre (381 (95% CI 339–428) vs. 713 (95% CI 641–792), P < 0.001), while there was no significant difference in the GMT between H3N2-inter and H3N2-pre. Since A(H1N1) predominated the 2018–2019 winter influenza epidemic, our results corroborated the epidemic subtype.

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Comparison of Human H3N2 Antibody Responses Elicited by Egg-Based, Cell-Based, and Recombinant Protein–Based Influenza Vaccines During the 2017–2018 Season

Clinical Infectious Diseases ◽

10.1093/cid/ciz996 ◽

2019 ◽

Vol 71 (6) ◽

pp. 1447-1453 ◽

Cited By ~ 1

Author(s):

Sigrid Gouma ◽

Seth J Zost ◽

Kaela Parkhouse ◽

Angela Branche ◽

David J Topham ◽

...

Keyword(s):

Recombinant Protein ◽

Neutralizing Antibody ◽

Antibody Responses ◽

Influenza Vaccines ◽

Vaccine Antigen ◽

H3n2 Virus ◽

High Dose ◽

Wild Type ◽

H1n1 Vaccine ◽

Antibody Titers

Abstract Background The H3N2 component of egg-based 2017–2018 influenza vaccines possessed an adaptive substitution that alters antigenicity. Several influenza vaccines include antigens that are produced through alternative systems, but a systematic comparison of different vaccines used during the 2017–2018 season has not been completed. Methods We compared antibody responses in humans vaccinated with Fluzone (egg-based, n = 23), Fluzone High-Dose (egg-based, n = 16), Flublok (recombinant protein–based, n = 23), or Flucelvax (cell-based, n = 23) during the 2017–2018 season. We completed neutralization assays using an egg-adapted H3N2 virus, a cell-based H3N2 virus, wild-type 3c2.A and 3c2.A2 H3N2 viruses, and the H1N1 vaccine strain. We also performed enzyme-linked immunosorbent assays using a recombinant wild-type 3c2.A hemagglutinin. Antibody responses were compared in adjusted analysis. Results Postvaccination neutralizing antibody titers to 3c2.A and 3c2.A2 were higher in Flublok recipients compared with Flucelvax or Fluzone recipients (P < .01). Postvaccination titers to 3c2.A and 3c2.A2 were similar in Flublok and Fluzone High-Dose recipients, though seroconversion rates trended higher in Flublok recipients. Postvaccination titers in Flucelvax recipients were low to all H3N2 viruses tested, including the cell-based H3N2 strain. Postvaccination neutralizing antibody titers to H1N1 were similar among the different vaccine groups. Conclusions These data suggest that influenza vaccine antigen match and dose are both important for eliciting optimal H3N2 antibody responses in humans. Future studies should be designed to determine if our findings directly impact vaccine effectiveness. Clinical Trials Registration NCT03068949.

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Disulfide bond formation during the folding of influenza virus hemagglutinin.

The Journal of Cell Biology ◽

10.1083/jcb.118.2.227 ◽

1992 ◽

Vol 118 (2) ◽

pp. 227-244 ◽

Cited By ~ 57

Author(s):

M S Segal ◽

J M Bye ◽

J F Sambrook ◽

M J Gething

Keyword(s):

Influenza Virus ◽

Disulfide Bonds ◽

Intracellular Transport ◽

Cellular Protein ◽

Cysteine Residues ◽

Mutant Proteins ◽

Influenza Virus Hemagglutinin ◽

Stalk Domain ◽

Do So

To study the importance of individual sulfhydryl residues during the folding and assembly in vivo of influenza virus hemagglutinin (HA), we have constructed and expressed a series of mutant HA proteins in which cysteines involved in three disulfide bonds have been substituted by serine residues. Investigations of the structure and intracellular transport of the mutant proteins indicate that (a) cysteine residues in the ectodomain are essential both for efficient folding of HA and for stabilization of the folded molecule; (b) cysteine residues in the globular portion of the ectodomain are likely to form native disulfide bonds rapidly and directly, without involvement of intermediate, nonnative linkages; and (c) cysteine residues in the stalk portion of the ectodomain also appear not to form intermediate disulfide bonds, even though they have the opportunity to do so, being separated from their correct partners by hundreds of amino acids including two or more other sulfhydryl residues. We propose a role for the cellular protein BiP in shielding the cysteine residues of the stalk domain during the folding process, thus preventing them from forming intermediate, nonnative disulfide bonds.

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