Hemagglutinin Stem Domain Research Articles

Cross-Reactive Fc-Mediated Antibody Responses to Influenza HA Stem Region in Human Sera Following Seasonal Vaccination

Background: Current influenza A vaccines primarily induce neutralizing antibodies targeting the variable hemagglutinin (HA) head domain, limiting their effectiveness against diverse or emerging influenza A virus (IAV) subtypes. The conserved HA stem domain, particularly the long α-helix (LAH) epitope, is a focus of universal vaccine research due to its cross-protective potential. Additionally, Fc-mediated functions such as antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP) are recognized as important protective immune mechanisms. This study evaluated IgG responses to the HA head, stem, and LAH regions and assessed cross-reactive potential through neutralization, ADCC, and ADCP assays. Methods: IgG responses to the HA head, stem, and LAH regions were measured in vaccinated individuals. Functional assays were conducted for neutralization, ADCC, and ADCP to evaluate the association between antibody levels and immune function. Results: The results showed that HA head-specific IgG increased significantly after vaccination in 50 individuals, whereas stem-specific IgG increased by 72% and LAH-specific IgG by 12–14%. Among the induced antibody subclasses, IgG1 was predominantly increased. Neutralization titers were detected in viruses of the same strain as the vaccine strain, but not in classical or pandemic strains (H5N1, H7N9). HA stem-specific IgG1 antibody titers showed a significant correlation with ADCC/ADCP activity breadth, but no correlation was observed with neutralization breadth. Conclusion: These findings suggest that although current influenza vaccines can induce HA stem-targeted cross-reactive antibodies, their quantity may be insufficient for broad cross-protection, underscoring the need for improved vaccine strategies.

Inserting CTL Epitopes of the Viral Nucleoprotein to Improve Immunogenicity and Protective Efficacy of Recombinant Protein against Influenza A Virus.

Conserved influenza virus proteins, such as the hemagglutinin stem domain (HA2), nucleoprotein (NP), and matrix protein (M), are the main targets in the development of universal influenza vaccines. Previously, we constructed a recombinant vaccine protein Flg-HA2-2-4M2ehs containing the extracellular domain of the M2 protein (M2e) and the aa76-130 sequence of the second HA subunit as target antigens. It demonstrated immunogenicity and broad protection against influenza A viruses after intranasal and parenteral administration. This study shows that CD8+ epitopes of NP, inserted into a flagellin-fused protein carrying M2e and HA2, affect the post-vaccination immune humoral response to virus antigens without reducing protection. No differences were found between the two proteins in their ability to stimulate the formation of follicular Th in the spleen, which may contribute to a long-lasting antigen-specific humoral response. The data obtained on Balb/c mice suggest that the insertion of CTL NP epitopes into the flagellin-fused protein carrying M2e and HA2 reduces the antibody response to M2e and A/H3N2. In C57Bl6 mice, this stimulates the formation of NP-specific CD8+ Tem and virus-specific mono- and multifunctional CD4+ and CD8+ Tem in the spleen and completely protects mice from influenza virus subtypes A/H1N1pdm09 and A/H3N2.

Rapid synthesis and screening of natively paired antibodies against influenza hemagglutinin stem via oPool+ display.

Antibody discovery is crucial for developing therapeutics and vaccines as well as understanding adaptive immunity. However, the lack of approaches to synthesize antibodies with defined sequences in a high-throughput manner represents a major bottleneck in antibody discovery. Here, we presented oPool+ display, which combines oligo pool synthesis and mRNA display to construct and characterize many natively paired antibodies in parallel. As a proof-of-concept, we applied oPool+ display to rapidly screen the binding activity of >300 natively paired influenza hemagglutinin (HA) antibodies against the conserved HA stem domain. Structural analysis of 16.ND.92, one of the identified HA stem antibodies, revealed a unique binding mode distinct from other known broadly neutralizing HA stem antibodies with convergent sequence features. Yet, despite such differences, 16.ND.92 remained broadly reactive and conferred in vivo protection. Overall, this study not only established an experimental platform that can be applied in both research and therapeutics to accelerate antibody discovery, but also provides molecular insights into antibody responses to the influenza HA stem, which is a major target for universal influenza vaccine development.

Anti-hemagglutinin monomeric nanobody provides prophylactic immunity against H1 subtype influenza A viruses.

Influenza viruses constitute a major threat to human health globally. The viral surface glycoprotein hemagglutinin (HA) is the immunodominant antigen, contains the site for binding to the cellular receptor (RBS), and it is the major target of neutralizing antibody responses post-infection. We developed llama-derived single chain antibody fragments (VHHs) specific for type A influenza virus. Four VHHs were identified and further characterized. VHH D81 bound residues in the proximity of the C-terminal region of HA1 of H1 and H5 subtypes, and showed weak neutralizing activity, whereas VHH B33 bound residues in the proximity of the N-terminal region of the HA's stem domain (HA2) of H1, H5, and H9 subtypes, and showed no neutralizing activity. Of most relevance, VHHs E13 and G41 recognized highly conserved conformational epitopes on the H1 HA's globular domain (HA1) and showed high virus neutralizing activity (ranging between 0.94 to 0.01μM), when tested against several human H1N1 isolates. Additionally, E13 displayed abrogated virus replication of a panel of H1N1 strains spanning over 80 years of antigenic drift and isolated from human, avian, and swine origin. Interestingly, E13 conferred protection in vivo at a dose as low as 0.05 mg/kg. Mice treated with E13 intranasally resulted in undetectable virus challenge loads in the lungs at day 4 post-challenge. The transfer of sterilizing pan-H1 immunity, by a dose in the range of micrograms given intranasally, is of major significance for a monomeric VHH and supports the further development of E13 as an immunotherapeutic agent for the mitigation of influenza infections.

Self-adjuvant multiepitope nanovaccine based on ferritin induced long-lasting and effective mucosal immunity against H3N2 and H1N1 viruses in mice

The influenza A virus (IAV) is a ubiquitous and continuously evolving respiratory pathogen. The intranasal vaccination mimicking natural infections is an attractive strategy for controlling IAVs. Multiepitope vaccines accurately targeting multiple conserved domains have the potential to broaden the protective scope of current seasonal influenza vaccines and reduce the risk of generating escape mutants. Here, multiple linear epitopes from the matrix protein 2 ectodomain (M2e) and the hemagglutinin stem domain (HA2) are fused with the Helicobacter pylori ferritin, a self-assembled nanocarrier and mucosal adjuvant, to develop a multiepitope nanovaccine. Through intranasal delivery, the prokaryotically expressed multiepitope nanovaccine elicits long-lasting mucosal immunity, broad humoral immunity, and robust cellular immunity without any adjuvants, and confers complete protection against H3N2 and H1N1 subtypes of IAV in mice. Importantly, this intranasal multiepitope nanovaccine triggers memory B-cell responses, resulting in secretory immunoglobulin A (sIgA) and serum immunoglobulin G (IgG) levels persisting for more than five months post-immunization. Therefore, this intranasal ferritin–based multiepitope nanovaccine represents a promising approach to combating respiratory pathogens.

Protective efficacy of a universal influenza mRNA vaccine against the challenge of H1 and H5 influenza A viruses in mice.

Current influenza vaccines need to be updated annually owing to constant antigenic drift in the globular head of the viral surface hemagglutinin (HA) glycoprotein. The immunogenic subdominant stem domain of HA is highly conserved and can be recognized by antibodies capable of binding multiple HA subtypes. Therefore, the HA stem antigen is a promising target for the design of universal influenza vaccines. On the basis of an established lipid nanoparticle-encapsulated mRNA vaccine platform, we designed and developed a novel universal influenza mRNA vaccine (mHAs) encoding the HA stem antigen of the influenza A (H1N1) virus. We tested the efficacy of the mHAs vaccine using a mouse model. The vaccine induced robust humoral and specific cellular immune responses against the stem region of HA. Importantly, two doses of the mHAs vaccine fully protected mice from lethal challenges of the heterologous H1N1 and heterosubtypic H5N8 influenza viruses. Vaccinated mice had less pathological lung damage and lower viral titers than control mice. These results suggest that an mRNA vaccine using the conserved stem region of HA may provide effective protection against seasonal and other possible influenza variants.

Epistasis reduces fitness costs of influenza A virus escape from stem-binding antibodies

The hemagglutinin (HA) stem region is a major target of universal influenza vaccine efforts owing to the presence of highly conserved epitopes across multiple influenza A virus (IAV) strains and subtypes. To explore the potential impact of vaccine-induced immunity targeting the HA stem, we examined the fitness effects of viral escape from stem-binding broadly neutralizing antibodies (stem-bnAbs). Recombinant viruses containing each individual antibody escape substitution showed diminished replication compared to wild-type virus, indicating that stem-bnAb escape incurred fitness costs. A second-site mutation in the HA head domain (N129D; H1 numbering) reduced the fitness effects observed in primary cell cultures and likely enabled the selection of escape mutations. Functionally, this putative permissive mutation increased HA avidity for its receptor. These results suggest a mechanism of epistasis in IAV, wherein modulating the efficiency of attachment eases evolutionary constraints imposed by the requirement for membrane fusion. Taken together, the data indicate that viral escape from stem-bnAbs is costly but highlights the potential for epistatic interactions to enable evolution within the functionally constrained HA stem domain.

Comparative Immunogenicity of Bacterially Expressed Soluble Trimers and Nanoparticle Displayed Influenza Hemagglutinin Stem Immunogens.

Current influenza vaccines need to be updated annually due to mutations in the globular head of the viral surface protein, hemagglutinin (HA). To address this, vaccine candidates have been designed based on the relatively conserved HA stem domain and have shown protective efficacy in animal models. Oligomerization of the antigens either by fusion to oligomerization motifs or display on self-assembling nanoparticle scaffolds, can induce more potent immune responses compared to the corresponding monomeric antigen due to multivalent engagement of B-cells. Since nanoparticle display can increase manufacturing complexity, and often involves one or more mammalian cell expressed components, it is important to characterize and compare various display and oligomerization scaffolds. Using a structure guided approach, we successfully displayed multiple copies of a previously designed soluble, trimeric influenza stem domain immunogen, pH1HA10, on the ferritin like protein, MsDps2 (12 copies), Ferritin (24 copies) and Encapsulin (180 copies). All proteins were expressed in Escherichia coli. The nanoparticle fusion immunogens were found to be well folded and bound to the influenza stem directed broadly neutralizing antibodies with high affinity. An 8.5 Å Cryo-EM map of Msdps2-pH1HA10 confirmed the successful design of the nanoparticle fusion immunogen. Mice immunization studies with the soluble trimeric stem and nanoparticle fusion constructs revealed that all of them were immunogenic, and protected mice against homologous (A/Belgium/145-MA/2009) and heterologous (A/Puerto Rico/8/1934) challenge with 10MLD50 mouse adapted virus. Although nanoparticle display conferred a small but statistically significant improvement in protection relative to the soluble trimer in a homologous challenge, heterologous protection was similar in both nanoparticle-stem immunized and trimeric stem immunized groups. Such rapidly producible, bacterially expressed antigens and nanoparticle scaffolds are useful modalities to tackle future influenza pandemics.

Influenza vaccination strategies targeting the hemagglutinin stem region.

Influenza is one of the best examples of highly mutable viruses that are able to escape immune surveillance. Indeed, in response to influenza seasonal infection or vaccination, the majority of the induced antibodies are strain‐specific. Current vaccine against the seasonal strains with the strategy of surveillance‐prediction‐vaccine does not cover an unmet virus strain leading to pandemic. Recently, antibodies targeting conserved epitopes on the hemagglutinin (HA) protein have been identified, albeit rarely, and they often showed broad protection. These antibody discoveries have brought the feasibility to develop a universal vaccine. Most of these antibodies bind the HA stem domain and accumulate in the memory B cell compartment. Broadly reactive stem‐biased memory responses were induced by infection with antigenically divergent influenza strains and were able to eradicate these viruses, together indicating the importance of generating memory B cells expressing high‐quality anti‐stem antibodies. Here, we emphasize recent progress in our understanding of how such memory B cells can be generated and discuss how these advances may be relevant to the quest for a universal influenza vaccine.

Induction of Cross-Reactive and Protective Antibody Responses After DNA Vaccination With MHCII-Targeted Stem Domain From Influenza Hemagglutinin.

Novel and more broadly protective vaccines against influenza are needed to efficiently meet antigenic drift and shift. Relevant to this end, the stem domain of hemagglutinin (HA) is highly conserved, and antibodies specific for epitopes located to the stem have been demonstrated to be able to confer broad protection against various influenza subtypes. However, a remaining challenge is to induce antibodies against the poorly immunogenic stem by vaccination strategies that can be scaled up for prophylactic vaccination of the general population. Here, we have developed DNA vaccines where the conserved stem domain of HA from influenza A/PR/8/34 (H1N1) and A/Shanghai/2/2013 (H7N9) was targeted toward MHC class II molecules on antigen-presenting cells (APC) for increased immunogenicity. Each of these vaccines induced antibodies that cross-reacted with other subtypes in the corresponding phylogenetic influenza groups. Importantly, when mixing the MHCII-targeted stem domains from H1N1 and H7N9 influenza viruses into one vaccine bolus, we observed broad protection against candidate stains from both phylogenetic groups 1 and 2.

Antibody-dependent enhancement of influenza disease promoted by increase in hemagglutinin stem flexibility and virus fusion kinetics

Several next-generation (universal) influenza vaccines and broadly neutralizing antibodies (bNAbs) are in clinical development. Some of these mediate inhibitions of virus replication at the postentry stage or use Fc-dependent mechanisms. Nonneutralizing antibodies have the potential to mediate enhancement of viral infection or disease. In the current study, two monoclonal antibodies (MAbs) 72/8 and 69/1, enhanced respiratory disease (ERD) in mice following H3N2 virus challenge by demonstrating increased lung pathology and changes in lung cytokine/chemokine levels. MAb 78/2 caused changes in the lung viral loads in a dose-dependent manner. Both MAbs increased HA sensitivity to trypsin cleavage at a higher pH range, suggesting MAb-induced conformational changes. pHrodo-labeled virus particles' entry and residence time in the endocytic compartment were tracked during infection of Madin-Darby canine kidney (MDCK) cells. Both MAbs reduced H3N2 virus residence time in the endocytic pathway, suggesting faster virus fusion kinetics. Structurally, 78/2 and 69/1 Fabs bound the globular head or base of the head domain of influenza hemagglutinin (HA), respectively, and induced destabilization of the HA stem domain. Together, this study describes Mab-induced destabilization of the influenza HA stem domain, faster kinetics of influenza virus fusion, and ERD in vivo. The in vivo animal model and in vitro assays described could augment preclinical safety evaluation of antibodies and next-generation influenza vaccines that generate antibodies which do not block influenza virus-receptor interaction.

Protect, modify, deprotect (PMD): A strategy for creating vaccines to elicit antibodies targeting a specific epitope

In creating vaccines against infectious agents, there is often a desire to direct an immune response toward a particular conformational epitope on an antigen. We present a method, called protect, modify, deprotect (PMD), to generate immunogenic proteins aimed to direct a vaccine-induced antibody (Ab) response toward an epitope defined by a specific monoclonal Ab (mAb). The mAb is used to protect the target epitope on the protein. Then the remaining exposed surfaces of the protein are modified to render them nonimmunogenic. Finally, the epitope is deprotected by removal of the mAb. The resultant protein is modified at surfaces other than the target epitope. We validate PMD using a well-characterized antigen, hen egg white lysozyme, then demonstrate the utility of PMD using influenza virus hemagglutinin (HA). We use an mAb to protect a highly conserved epitope on the stem domain of HA. Exposed surface amines are then modified with short polyethylene glycol chains. The resultant antigen shows markedly reduced binding to mAbs that target the head region of HA, while maintaining binding to mAbs at the epitope of interest. This antigenic preference is also observed with yeast cells displaying Ab fragments. Antisera from guinea pigs immunized with the PMD-modified HA show increased cross-reactivity with HAs from other influenza strains, compared with antisera obtained with unmodified HA trimers. PMD has the potential to direct an Ab response at high resolution and could be used in combination with other such strategies. There are many attractive targets for the application of PMD.

Immunodominance and Antigenic Variation of Influenza Virus Hemagglutinin: Implications for Design of Universal Vaccine Immunogens.

Influenza viruses routinely acquire mutations in their hemagglutinin (HA) and neuraminidase (NA) glycoproteins that abrogate binding of pre-existing antibodies in a process known as antigenic drift. Most human antibodies against HA and NA are directed against epitopes that are hypervariable and not against epitopes that are conserved among different influenza virus strains. Universal influenza vaccines are currently being developed to elicit protective responses against functionally conserved sites on influenza proteins where viral escape mutations can result in large fitness costs [1]. Universal vaccine targets include the highly conserved HA stem domain [2-12], the less conserved HA receptor-binding site (RBS) [13-16], as well as conserved sites on NA [17-19]. One central challenge of universal vaccine efforts is to steer human antibody responses away from immunodominant, variable epitopes and towards subdominant, functionally conserved sites. Overcoming this challenge will require further understanding of the structural basis of broadly neutralizing HA and NA antibody binding epitopes and factors that influence immunodominance hierarchies of human antibody responses.

Lowered pH Leads to Fusion Peptide Release and a Highly Dynamic Intermediate of Influenza Hemagglutinin.

Hemagglutinin (HA), the membrane-bound fusion protein of the influenza virus, enables the entry of virus into host cells via a structural rearrangement. There is strong evidence that the primary trigger for this rearrangement is the low pH environment of a late endosome. To understand the structural basis and the dynamic consequences of the pH trigger, we employed explicit-solvent molecular dynamics simulations to investigate the initial stages of the HA transition. Our results indicate that lowered pH destabilizes HA and speeds up the dissociation of the fusion peptides (FPs). A buried salt bridge between the N-terminus and Asp1122 of HA stem domain locks the FPs and may act as one of the pH sensors. In line with recent observations from simplified protein models, we find that, after the dissociation of FPs, a structural order-disorder transition in a loop connecting the central coiled-coil to the C-terminal domains produces a highly mobile HA. This motion suggests the existence of a long-lived asymmetric or "symmetry-broken" intermediate during the HA conformational change. This intermediate conformation is consistent with models of hemifusion, and its early formation during the conformational change has implications for the aggregation seen in HA activity.

IGHV1-69 polymorphism modulates anti-influenza antibody repertoires, correlates with IGHV utilization shifts and varies by ethnicity.

IGHV polymorphism provides a rich source of humoral immune system diversity. One important example is the IGHV1-69 germline gene where the biased use of alleles that encode the critical CDR-H2 Phe54 (F-alleles) to make broadly neutralizing antibodies (HV1-69-sBnAb) to the influenza A hemagglutinin stem domain has been clearly established. However, whether IGHV1-69 polymorphism can also modulate B cell function and Ab repertoire expression through promoter and copy number (CN) variations has not been reported, nor has whether IGHV1-69 allelic distribution is impacted by ethnicity. Here we studied a cohort of NIH H5N1 vaccinees and demonstrate for the first time the influence of IGHV1-69 polymorphism on V-segment usage, somatic hypermutation and B cell expansion that elucidates the dominance of F-alleles in HV1-69-sBnAbs. We provide evidence that Phe54/Leu54 (F/L) polymorphism correlates with shifted repertoire usage of other IGHV germline genes. In addition, we analyzed ethnically diverse individuals within the 1000 genomes project and discovered marked variations in F- and L- genotypes and CN among the various ethnic groups that may impact HV1-69-sBnAb responses. These results have immediate implications for understanding HV1-69-sBnAb responses at the individual and population level and for the design and implementation of “universal” influenza vaccine.

Hemagglutinin Sequence Conservation Guided Stem Immunogen Design from Influenza A H3 Subtype.

Seasonal epidemics caused by influenza A (H1 and H3 subtypes) and B viruses are a major global health threat. The traditional, trivalent influenza vaccines have limited efficacy because of rapid antigenic evolution of the circulating viruses. This antigenic variability mediates viral escape from the host immune responses, necessitating annual vaccine updates. Influenza vaccines elicit a protective antibody response, primarily targeting the viral surface glycoprotein hemagglutinin (HA). However, the predominant humoral response is against the hypervariable head domain of HA, thereby restricting the breadth of protection. In contrast, the conserved, subdominant stem domain of HA is a potential “universal” vaccine candidate. We designed an HA stem-fragment immunogen from the 1968 pandemic H3N2 strain (A/Hong Kong/1/68) guided by a comprehensive H3 HA sequence conservation analysis. The biophysical properties of the designed immunogen were further improved by C-terminal fusion of a trimerization motif, “isoleucine-zipper”, or “foldon”. These immunogens elicited cross-reactive, antiviral antibodies and conferred partial protection against a lethal, homologous HK68 virus challenge in vivo. Furthermore, bacterial expression of these immunogens is economical and facilitates rapid scale-up.

Protective immunity based on the conserved hemagglutinin stalk domain and its prospects for universal influenza vaccine development.

Influenza virus surface glycoprotein hemagglutinin (HA) is an excellent and chief target that elicits neutralizing antibodies during vaccination or natural infection. Its HA2 subunit (stem domain) is most conserved as compared to HA1 subunit (globular head domain). Current influenza vaccine relies on globular head domain that provides protection only against the homologous vaccine strains, rarely provides cross-protection against divergent strains, and needs to be updated annually. There is an urge for a truly universal vaccine that provides broad cross-protection against different subtype influenza A viruses along with influenza B viruses and need not be updated annually. Antibodies against the stem domain of hemagglutinin (HA) are able to neutralize a wide spectrum of influenza virus strains and subtypes. These stem-specific antibodies have great potential for the development of universal vaccine against influenza viruses. In this review, we have discussed the stem-specific cross-reactive antibodies and heterosubtypic protection provided by them. We have also discussed their epitope-based DNA vaccine and their future prospects in this scenario.

Distribution of surface glycoproteins on influenza A virus determined by electron cryotomography

We use electron cryotomography to reconstruct virions of two influenza A H3N2 virus strains. The maps reveal the structure of the viral envelope containing hemagglutinin (HA) and neuraminidase (NA) glycoproteins and the virus interior containing a matrix layer and an assembly of ribonucleoprotein particles (RNPs) that package the genome. We build a structural model for the viral surface by locating copies of the X-ray structure of the HA ectodomain into density peaks on the virus surface. We calculate inter-glycoprotein distances and the fractional volume occupied by glycoproteins. The models suggest that for typical HA densities on virus, Fabs can bind to epitopes on the HA stem domain. The models also show how membrane curvature may influence the number of glycoproteins that can simultaneously interact with a target surface of receptors.

Heterosubtypic Neutralizing Monoclonal Antibodies Cross-Protective against H5N1 and H1N1 Recovered from Human IgM+ Memory B Cells

BackgroundThe hemagglutinin (HA) glycoprotein is the principal target of protective humoral immune responses to influenza virus infections but such antibody responses only provide efficient protection against a narrow spectrum of HA antigenic variants within a given virus subtype. Avian influenza viruses such as H5N1 are currently panzootic and pose a pandemic threat. These viruses are antigenically diverse and protective strategies need to cross protect against diverse viral clades. Furthermore, there are 16 different HA subtypes and no certainty the next pandemic will be caused by an H5 subtype, thus it is important to develop prophylactic and therapeutic interventions that provide heterosubtypic protection.Methods and FindingsHere we describe a panel of 13 monoclonal antibodies (mAbs) recovered from combinatorial display libraries that were constructed from human IgM+ memory B cells of recent (seasonal) influenza vaccinees. The mAbs have broad heterosubtypic neutralizing activity against antigenically diverse H1, H2, H5, H6, H8 and H9 influenza subtypes. Restriction to variable heavy chain gene IGHV1-69 in the high affinity mAb panel was associated with binding to a conserved hydrophobic pocket in the stem domain of HA. The most potent antibody (CR6261) was protective in mice when given before and after lethal H5N1 or H1N1 challenge.ConclusionsThe human monoclonal CR6261 described in this study could be developed for use as a broad spectrum agent for prophylaxis or treatment of human or avian influenza infections without prior strain characterization. Moreover, the CR6261 epitope could be applied in targeted vaccine strategies or in the design of novel antivirals. Finally our approach of screening the IgM+ memory repertoire could be applied to identify conserved and functionally relevant targets on other rapidly evolving pathogens.

A cell-based reglucosylation assay demonstrates the role of GT1 in the quality control of a maturing glycoprotein

The endoplasmic reticulum (ER) protein GT1 (UDP-glucose: glycoprotein glucosyltransferase) is the central enzyme that modifies N-linked carbohydrates based upon the properties of the polypeptide backbone of the maturing substrate. GT1 adds glucose residues to nonglucosylated proteins that fail the quality control test, supporting ER retention through persistent binding to the lectin chaperones calnexin and calreticulin. How GT1 functions in its native environment on a maturing substrate is poorly understood. We analyzed the reglucosylation of a maturing model glycoprotein, influenza hemagglutinin (HA), in the intact mammalian ER. GT1 reglucosylated N-linked glycans in the slow-folding stem domain of HA once the nascent chain was released from the ribosome. Maturation mutants that disrupted the oxidation or oligomerization of HA also supported region-specific reglucosylation by GT1. Therefore, GT1 acts as an ER quality control sensor by posttranslationally reglucosylating glycans on slow-folding or nonnative domains to recruit chaperones specifically to critical aberrant regions.