Librator, a platform for optimized sequence editing, design, and expression of influenza virus proteins

AbstractArtificial mutagenesis and chimeric/mosaic protein engineering have laid the foundation for antigenic characterization1 and universal vaccine design2–4 for influenza viruses. However, many methods used for influenza research and vaccine development require sequence editing and protein expression, limiting their applicability and the progress of related research to specialists. Rapid tools allowing even novice influenza researchers to properly analyze and visualize influenza protein sequences with accurate nomenclature are needed to expand the research field. To address this need, we developed Librator, a system for analyzing and designing protein sequences of influenza virus Hemagglutinin (HA) and Neuraminidase (NA). With Librator’s graphical user interface (GUI) and built-in sequence editing functions, biologists can easily analyze influenza sequences and phylogenies, automatically port sequences to visualize structures, then readily mutate target residues and design sequences for antigen probes and chimeric/mosaic proteins efficiently and accurately. This system provides optimized fragment design for Gibson Assembly5 of HA and NA expression constructs based on peptide conservation of all historical HA and NA sequences, ensuring fragments are reusable and compatible, allowing for significant reagent savings. Use of Librator will significantly facilitate influenza research and vaccine antigen design.

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Universal influenza virus neuraminidase vaccine elicits protective immune responses against human seasonal and pre-pandemic strains

Journal of Virology ◽

10.1128/jvi.00759-21 ◽

2021 ◽

Author(s):

Amanda L. Skarlupka ◽

Anne Gaelle Bebin-Blackwell ◽

Spencer F. Sumner ◽

Ted M. Ross

Keyword(s):

North Carolina ◽

Influenza Virus ◽

Vaccine Development ◽

Surface Protein ◽

Influenza Virus Vaccine ◽

Influenza Viruses ◽

Influenza Vaccines ◽

Vaccine Antigen ◽

Viet Nam ◽

H5n1 Influenza

The hemagglutinin (HA) surface protein is the primary immune target for most influenza vaccines. The neuraminidase (NA) surface protein is often a secondary target for vaccine designs. In this study, computationally optimized broadly reactive antigen methodology was used to generate the N1-I NA vaccine antigen that was designed to cross-react with avian, swine, and human influenza viruses of N1 NA subtype. The elicited antibodies bound to NA proteins derived from A/California/07/2009 (H1N1)pdm09, A/Brisbane/59/2007 (H1N1), A/Swine/North Carolina/154074/2015 (H1N1) and A/Viet Nam/1203/2004 (H5N1) influenza viruses, with NA-neutralizing activity against a broad panel of HXN1 influenza strains. Mice vaccinated with the N1-I COBRA NA vaccine were protected from mortality and viral lung titers were lower when challenged with four different viral challenges: A/California/07/2009, A/Brisbane/59/2007, A/Swine/North Carolina/154074/2015 and A/Viet Nam/1203/2004. Vaccinated mice had little to no weight loss against both homologous, but also cross-NA genetic clade challenges. Lung viral titers were lower compared to the mock vaccinated mice, and at times, equivalent to the homologous control. Thus, the N1-I COBRA NA antigen has the potential to be a complimentary component in a multi-antigen universal influenza virus vaccine formulation that also contains HA antigens. Importance The development and distribution of a universal influenza vaccines would alleviate global economic and public health stress from annual influenza virus outbreaks. The influenza virus NA vaccine antigen allows for protection from multiple HA subtypes and virus host origins, but it has not been the focus of vaccine development. The N1-I NA antigen described here protected mice from direct challenge of four distinct influenza viruses and inhibited the enzymatic activity of a N1 influenza virus panel. The use of the NA antigen in combination with the HA widens the breadth of protection against various virus strains. Therefore, this research opens the door to the development of a longer lasting vaccine with increased protective breadth.

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Influenza–Host Interplay and Strategies for Universal Vaccine Development

Vaccines ◽

10.3390/vaccines8030548 ◽

2020 ◽

Vol 8 (3) ◽

pp. 548

Author(s):

Hye Suk Hwang ◽

Mincheol Chang ◽

Yoong Ahm Kim

Keyword(s):

Influenza Virus ◽

Influenza A ◽

Vaccine Development ◽

Current Knowledge ◽

Host Cells ◽

Influenza A Viruses ◽

Universal Vaccine ◽

Lectin Receptors ◽

Endosomal Membrane ◽

Influenza Virus Hemagglutinin

Influenza is an annual epidemic and an occasional pandemic caused by pathogens that are responsible for infectious respiratory disease. Humans are highly susceptible to the infection mediated by influenza A viruses (IAV). The entry of the virus is mediated by the influenza virus hemagglutinin (HA) glycoprotein that binds to the cellular sialic acid receptors and facilitates the fusion of the viral membrane with the endosomal membrane. During IAV infection, virus-derived pathogen-associated molecular patterns (PAMPs) are recognized by host intracellular specific sensors including toll-like receptors (TLRs), C-type lectin receptors, retinoic acid-inducible gene-I (RIG-I)-like receptors (RLRs), and nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs) either on the cell surface or intracellularly in endosomes. Herein, we comprehensively review the current knowledge available on the entry of the influenza virus into host cells and the molecular details of the influenza virus–host interface. We also highlight certain strategies for the development of universal influenza vaccines.

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Influenza Virus Hemagglutinin Glycoproteins with Different N-Glycan Patterns Activate Dendritic CellsIn Vitro

Journal of Virology ◽

10.1128/jvi.00452-16 ◽

2016 ◽

Vol 90 (13) ◽

pp. 6085-6096 ◽

Cited By ~ 18

Author(s):

Wen-Chun Liu ◽

Yu-Li Lin ◽

Maureen Spearman ◽

Pei-Yun Cheng ◽

Michael Butler ◽

...

Keyword(s):

Influenza Virus ◽

Immune Responses ◽

Vaccine Development ◽

Influenza Viruses ◽

Viral Escape ◽

Complex Type ◽

Adaptive Immune Responses ◽

Adaptive Immune ◽

Influenza Virus Hemagglutinin ◽

High Mannose

ABSTRACTInfluenza virus hemagglutinin (HA) N-glycans play important regulatory roles in the control of virus virulence, antigenicity, receptor-binding specificity, and viral escape from the immune response. Considered essential for controlling innate and adaptive immune responses against influenza virus infections, dendritic cells (DCs) trigger proinflammatory and adaptive immune responses in hosts. In this study, we engineered Chinese hamster ovary (CHO) cell lines expressing recombinant HA from pandemic H1, H5, and H7 influenza viruses. rH1HA, rH5HA, and rH7HA were obtained as wild-type proteins or in the presence of kifunensine (KIF) or further with endo-β-N-acetylglucosaminidase-treated KIF (KIF+E) to generate single-N-acetylglucosamine (GlcNAc) N-glycans consisting of (i) terminally sialylated complex-type N-glycans, (ii) high-mannose-type N-glycans, and (iii) single-GlcNAc-type N-glycans. Our results show that high-mannose-type and single-GlcNAc-type N-glycans, but not complex-type N-glycans, are capable of inducing more active hIL12 p40, hIL12 p70, and hIL-10 production in human DCs. Significantly higher HLA-DR, CD40, CD83, and CD86 expression levels, as well reduced endocytotic capacity in human DCs, were noted in the high-mannose-type rH1HA and single-GlcNAc-type rH1HA groups than in the complex-type N-glycan rH1HA group. Our data indicate that native avian rHA proteins (H5N1 and H7N9) are more immunostimulatory than human rHA protein (pH1N1). The high-mannose-type or single-GlcNAc-type N-glycans of both avian and human HA types are more stimulatory than the complex-type N-glycans. HA-stimulated DC activation was accomplished partially through a mannose receptor(s). These results provide more understanding of the contribution of glycosylation of viral proteins to the immune responses and may have implications for vaccine development.IMPORTANCEInfluenza viruses trigger seasonal epidemics or pandemics with mild-to-severe consequences for human and poultry populations. DCs are the most potent professional antigen-presenting cells, which play a crucial role in the link between innate and adaptive immunity. In this study, we obtained stable-expression CHO cells to produce rH1HA, rH5HA, and rH7HA proteins containing distinct N-glycan patterns. These rHA proteins, each with a distinct N-glycan pattern, were used to investigate interactions with mouse and human DCs. Our data indicate that native avian rHA proteins (H5N1 and H7N9) are more immunostimulatory than human rHA protein (pH1N1). High-mannose-type and single-GlcNAc-type N-glycans were more effective than complex-type N-glycans in triggering mouse and human DC activation and maturation. We believe these results provide some useful information for influenza vaccine development regarding how influenza virus HA proteins with different types of N-glycans activate DCs.

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Original antigenic sin priming of influenza virus hemagglutinin stalk antibodies

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1920321117 ◽

2020 ◽

Vol 117 (29) ◽

pp. 17221-17227 ◽

Cited By ~ 3

Author(s):

Claudia P. Arevalo ◽

Valerie Le Sage ◽

Marcus J. Bolton ◽

Theresa Eilola ◽

Jennifer E. Jones ◽

...

Keyword(s):

Influenza Virus ◽

Antibody Response ◽

Influenza A ◽

Influenza Viruses ◽

Antibody Responses ◽

Virus Infections ◽

Universal Vaccine ◽

Influenza Virus Hemagglutinin ◽

Stalk Domain ◽

Original Antigenic Sin

Immunity to influenza viruses can be long-lived, but reinfections with antigenically distinct viral strains and subtypes are common. Reinfections can boost antibody responses against viral strains first encountered in childhood through a process termed “original antigenic sin.” It is unknown how initial childhood exposures affect the induction of antibodies against the hemagglutinin (HA) stalk domain of influenza viruses. This is an important consideration since broadly reactive HA stalk antibodies can protect against infection, and universal vaccine platforms are being developed to induce these antibodies. Here we show that experimentally infected ferrets and naturally infected humans establish strong “immunological imprints” against HA stalk antigens first encountered during primary influenza virus infections. We found that HA stalk antibodies are surprisingly boosted upon subsequent infections with antigenically distinct influenza A virus subtypes. Paradoxically, these heterosubtypic-boosted HA stalk antibodies do not bind efficiently to the boosting influenza virus strain. Our results demonstrate that an individual’s HA stalk antibody response is dependent on the specific subtype of influenza virus that they first encounter early in life. We propose that humans are susceptible to heterosubtypic influenza virus infections later in life since these viruses boost HA stalk antibodies that do not bind efficiently to the boosting antigen.

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A Study on Host Tropism Determinants of Influenza Virus Using Machine Learning

Current Bioinformatics ◽

10.2174/1574893614666191104160927 ◽

2020 ◽

Vol 15 (2) ◽

pp. 121-134 ◽

Cited By ~ 2

Author(s):

Eunmi Kwon ◽

Myeongji Cho ◽

Hayeon Kim ◽

Hyeon S. Son

Keyword(s):

Machine Learning ◽

Amino Acids ◽

Influenza Virus ◽

Random Forest ◽

Physicochemical Properties ◽

Protein Sequences ◽

Influenza Viruses ◽

Host Tropism ◽

Post Hoc ◽

Ha Protein

Background: The host tropism determinants of influenza virus, which cause changes in the host range and increase the likelihood of interaction with specific hosts, are critical for understanding the infection and propagation of the virus in diverse host species. Methods: Six types of protein sequences of influenza viral strains isolated from three classes of hosts (avian, human, and swine) were obtained. Random forest, naïve Bayes classification, and knearest neighbor algorithms were used for host classification. The Java language was used for sequence analysis programming and identifying host-specific position markers. Results: A machine learning technique was explored to derive the physicochemical properties of amino acids used in host classification and prediction. HA protein was found to play the most important role in determining host tropism of the influenza virus, and the random forest method yielded the highest accuracy in host prediction. Conserved amino acids that exhibited host-specific differences were also selected and verified, and they were found to be useful position markers for host classification. Finally, ANOVA analysis and post-hoc testing revealed that the physicochemical properties of amino acids, comprising protein sequences combined with position markers, differed significantly among hosts. Conclusion: The host tropism determinants and position markers described in this study can be used in related research to classify, identify, and predict the hosts of influenza viruses that are currently susceptible or likely to be infected in the future.

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TMPRSS2 and TMPRSS4 Facilitate Trypsin-Independent Spread of Influenza Virus in Caco-2 Cells

Journal of Virology ◽

10.1128/jvi.00239-10 ◽

2010 ◽

Vol 84 (19) ◽

pp. 10016-10025 ◽

Cited By ~ 104

Author(s):

Stephanie Bertram ◽

Ilona Glowacka ◽

Paulina Blazejewska ◽

Elizabeth Soilleux ◽

Paul Allen ◽

...

Keyword(s):

Influenza Virus ◽

Serine Proteases ◽

Alveolar Epithelium ◽

Influenza Viruses ◽

Target Cells ◽

Human Respiratory Tract ◽

Proteolytic Activation ◽

Viral Spread ◽

Endogenous Expression ◽

Influenza Virus Hemagglutinin

ABSTRACT Proteolysis of influenza virus hemagglutinin by host cell proteases is essential for viral infectivity, but the proteases responsible are not well defined. Recently, we showed that engineered expression of the type II transmembrane serine proteases (TTSPs) TMPRSS2 and TMPRSS4 allows hemagglutinin (HA) cleavage. Here we analyzed whether TMPRSS2 and TMPRSS4 are expressed in influenza virus target cells and support viral spread in the absence of exogenously added protease (trypsin). We found that transient expression of TMPRSS2 and TMPRSS4 resulted in HA cleavage and trypsin-independent viral spread. Endogenous expression of TMPRSS2 and TMPRSS4 in cell lines correlated with the ability to support the spread of influenza virus in the absence of trypsin, indicating that these proteases might activate influenza virus in naturally permissive cells. Indeed, RNA interference (RNAi)-mediated knockdown of both TMPRSS2 and TMPRSS4 in Caco-2 cells, which released fully infectious virus without trypsin treatment, markedly reduced the spread of influenza virus, demonstrating that these proteases were responsible for efficient proteolytic activation of HA in this cell line. Finally, TMPRSS2 was found to be coexpressed with the major receptor determinant of human influenza viruses, 2,6-linked sialic acids, in human alveolar epithelium, indicating that viral target cells in the human respiratory tract express TMPRSS2. Collectively, our results point toward an important role for TMPRSS2 and possibly TMPRSS4 in influenza virus replication and highlight the former protease as a potential therapeutic target.

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Perforin and Fas Cytolytic Pathways Coordinately Shape the Selection and Diversity of CD8+-T-Cell Escape Variants of Influenza Virus

Journal of Virology ◽

10.1128/jvi.79.13.8545-8559.2005 ◽

2005 ◽

Vol 79 (13) ◽

pp. 8545-8559 ◽

Cited By ~ 18

Author(s):

Graeme E. Price ◽

Lei Huang ◽

Rong Ou ◽

Menghua Zhang ◽

Demetrius Moskophidis

Keyword(s):

Influenza Virus ◽

T Cell ◽

Vaccine Development ◽

Influenza Virus Infection ◽

Influenza Viruses ◽

Viral Escape ◽

Viral Control ◽

Ctl Escape ◽

Selection Of

ABSTRACT Antigenic variation is a viral strategy exploited to promote survival in the face of the host immune response and represents a major challenge for efficient vaccine development. Influenza viruses are pathogens with high transmissibility and mutation rates, enabling viral escape from immunity induced by prior infection or vaccination. Intense selection from neutralizing antibody drives antigenic changes in the surface glycoproteins, resulting in emergence of new strains able to reinfect hosts immune to previously circulating viruses. CD8+ cytotoxic T cells (CTLs) also provide protective immunity from influenza virus infection and may contribute to the antigenic evolution of influenza viruses. Utilizing mice transgenic for an influenza virus NP366-374 peptide-specific T-cell receptor, we demonstrated that the respiratory tract is a suitable site for generation of escape variants of influenza virus selected by CTL in vivo. In this report the contributions of the perforin and Fas pathways utilized by influenza virus-specific CTLs in viral clearance and selection of CTL escape variants have been evaluated. While transgenic CTLs deficient in either perforin- or Fas-mediated pathways are efficient in initial pulmonary viral control, variant virus emergence was observed in all the mice studied, although the spectrum of viral CTL escape variants selected varied profoundly. Thus, a less-restricted repertoire of escape variants was observed in mice with an intact perforin cytotoxic pathway compared with a limited variant diversity in perforin pathway-deficient mice, although maximal variant diversity was observed in mice having both Fas and perforin pathways intact. We conclude that selection of viral CTL escape variants reflects coordinate action between the tightly controlled perforin/granzyme pathway and the more promiscuous Fas/FasL pathway.

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A recombinant protein containing influenza viral conserved epitopes and superantigen induces broad-spectrum protection

10.1101/2021.07.08.451596 ◽

2021 ◽

Author(s):

Yansheng Li ◽

Mingkai Xu ◽

Yongqiang Li ◽

Wu Gu ◽

Gulinare Halimu ◽

...

Keyword(s):

T Cells ◽

Influenza Virus ◽

Recombinant Protein ◽

Broad Spectrum ◽

Influenza Pandemic ◽

Influenza Virus Infection ◽

Influenza Viruses ◽

Lung Damage ◽

Potential Candidate ◽

Universal Vaccine

Influenza pandemic poses public health threats annually for lacking vaccine which provides cross-protection against novel and emerging influenza viruses. Combining conserved antigens inducing cross-protective antibody response with epitopes activating cross-protective cytotoxic T-cells would offer an attractive strategy for developing universal vaccine. In this study, we constructed a recombinant protein NMHC consisting of influenza viral conserved epitopes and superantigen fragment. NMHC promoted the mature of bone marrow-derived dendritic cells and induced CD4+ T cells to differentiate into Th1, 32 Th2 and Th17 subtypes. Mice vaccinated with NMHC produced high level of immunoglobulins which cross-bound to HA fragments from six influenza virus subtypes with high antibody titers. Anti-NMHC serum showed potent hemagglutinin inhibition effects to highly divergent group 1 (H1 subtypes) and group 2 (H3 subtype) influenza virus strains. And purified anti-NMHC antibodies could bind to multiple HAs with high affinities. NMHC vaccination effectively protected the mice from infection and lung damage challenged by two subtypes of H1N1 influenza virus. Moreover, NMHC vaccination elicited CD4+ and CD8+ T-cell responses to clear the virus from infected tissue and prevent virus spreading. In conclusion, this study provided proof of concept for triggering both B cells and T cells immune responses against multiple influenza virus infection, and NMHC may be a potential candidate of universal broad-spectrum vaccine for various influenza virus prevention and therapy.

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A recombinant protein containing influenza viral conserved epitopes and superantigen induces broad-spectrum protection

eLife ◽

10.7554/elife.71725 ◽

2021 ◽

Vol 10 ◽

Author(s):

Yansheng Li ◽

Mingkai Xu ◽

Yongqiang Li ◽

Wu Gu ◽

Gulinare Halimu ◽

...

Keyword(s):

Influenza Virus ◽

T Cell ◽

Recombinant Protein ◽

Broad Spectrum ◽

T Cell Responses ◽

H1n1 Influenza ◽

Influenza Viruses ◽

Lung Damage ◽

Universal Vaccine ◽

Cell Responses

Influenza pandemics pose public health threats annually for lacking vaccine which provides cross-protection against novel and emerging influenza viruses. Combining conserved antigens that induce cross-protective antibody responses with epitopes that activate cross-protective T cell responses might be an attractive strategy for developing a universal vaccine. In this study, we constructed a recombinant protein named NMHC which consist of influenza viral conserved epitopes and a superantigen fragment. NMHC promoted the maturation of bone marrow-derived dendritic cells and induced CD4+ T cells to differentiate into Th1, Th2, and Th17 subtypes. Mice vaccinated with NMHC produced high levels of immunoglobulins that cross-bound to HA fragments from six influenza virus subtypes with high antibody titers. Anti-NMHC serum showed potent hemagglutinin inhibition effects to highly divergent group 1 (H1 subtype) and group 2 (H3 subtype) influenza virus strains. Furthermore, purified anti-NMHC antibodies bound to multiple HAs with high affinities. NMHC vaccination effectively protected mice from infection and lung damage when exposed to two subtypes of H1N1 influenza virus. Moreover, NMHC vaccination elicited CD4+ and CD8+ T cell responses that cleared the virus from infected tissues and prevented virus spread. In conclusion, this study provides proof of concept that NMHC vaccination triggers B and T cell immune responses against multiple influenza virus infections. Therefore, NMHC might be a candidate universal broad-spectrum vaccine for the prevention and treatment of multiple influenza viruses.

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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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