Viral Subpopulation Screening Guides in Designing a High Interferon-Inducing Live Attenuated Influenza Vaccine by Targeting Rare Mutations in NS1 and PB2 Proteins

ABSTRACTInfluenza A viruses continue to circulate among wild birds and poultry worldwide, posing constant pandemic threats to humans. Effective control of emerging influenza viruses requires new broadly protective vaccines. Live attenuated influenza vaccines with truncations in nonstructural protein 1 (NS1) have shown broad protective efficacies in birds and mammals, which correlate with the ability to induce elevated interferon responses in the vaccinated hosts. Given the extreme diversity of influenza virus populations, we asked if we could improve an NS1-truncated live attenuated influenza vaccine developed for poultry (PC4) by selecting viral subpopulations with enhanced interferon-inducing capacities. Here, we deconstructed a de novo population of PC4 through plaque isolation, created a large library of clones, and assessed their interferon-inducing phenotypes. While most of the clones displayed the parental interferon-inducing phenotype in cell culture, few clones showed enhanced interferon-inducing phenotypes in cell culture and chickens. The enhanced interferon-inducing phenotypes were linked to either a deletion in NS1 (NS1Δ76-86) or a substitution in polymerase basic 2 protein (PB2-D309N). The NS1Δ76-86 deletion disrupted the putative eukaryotic translation initiation factor 4GI-binding domain and promoted the synthesis of biologically active interferons. The PB2-D309N substitution enhanced the early transcription of interferon mRNA, revealing a novel role for the 309D residue in suppression of interferon responses. We combined these mutations to engineer a novel vaccine candidate that induced additive amounts of interferons and stimulated protective immunity in chickens. Therefore, viral subpopulation screening approaches can guide the design of live vaccines with strong immunostimulatory properties.IMPORTANCE Effectiveness of NS1-truncated live attenuated influenza vaccines relies heavily on their ability to induce elevated interferon responses in vaccinated hosts. Influenza viruses contain diverse particle subpopulations with distinct phenotypes. We show that live influenza vaccines can contain underappreciated subpopulations with enhanced interferon-inducing phenotypes. The genomic traits of such virus subpopulations can be used to further improve the efficacy of the current live vaccines.

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A Live Attenuated Influenza Vaccine Elicits Enhanced Heterologous Protection When the Internal Genes of the Vaccine Are Matched to Those of the Challenge Virus

Journal of Virology ◽

10.1128/jvi.01065-19 ◽

2019 ◽

Vol 94 (4) ◽

Cited By ~ 3

Author(s):

Andrew Smith ◽

Laura Rodriguez ◽

Maya El Ghouayel ◽

Aitor Nogales ◽

Jeffrey M. Chamberlain ◽

...

Keyword(s):

Influenza Vaccine ◽

Influenza A ◽

H1n1 Virus ◽

Influenza Infection ◽

Influenza Viruses ◽

Influenza Vaccines ◽

Temperature Sensitive ◽

Live Attenuated Influenza Vaccine ◽

Cell Immunity

ABSTRACT Influenza A virus (IAV) causes significant morbidity and mortality, despite the availability of viral vaccines. The efficacy of live attenuated influenza vaccines (LAIVs) has been especially poor in recent years. One potential reason is that the master donor virus (MDV), on which all LAIVs are based, contains either the internal genes of the 1960 A/Ann Arbor/6/60 or the 1957 A/Leningrad/17/57 H2N2 viruses (i.e., they diverge considerably from currently circulating strains). We previously showed that introduction of the temperature-sensitive (ts) residue signature of the AA/60 MDV into a 2009 pandemic A/California/04/09 H1N1 virus (Cal/09) results in only 10-fold in vivo attenuation in mice. We have previously shown that the ts residue signature of the Russian A/Leningrad/17/57 H2N2 LAIV (Len LAIV) more robustly attenuates the prototypical A/Puerto Rico/8/1934 (PR8) H1N1 virus. In this work, we therefore introduced the ts signature from Len LAIV into Cal/09. This new Cal/09 LAIV is ts in vitro, highly attenuated (att) in mice, and protects from a lethal homologous challenge. In addition, when our Cal/09 LAIV with PR8 hemagglutinin and neuraminidase was used to vaccinate mice, it provided enhanced protection against a wild-type Cal/09 challenge relative to a PR8 LAIV with the same attenuating mutations. These findings suggest it may be possible to improve the efficacy of LAIVs by better matching the sequence of the MDV to currently circulating strains. IMPORTANCE Seasonal influenza infection remains a major cause of disease and death, underscoring the need for improved vaccines. Among current influenza vaccines, the live attenuated influenza vaccine (LAIV) is unique in its ability to elicit T-cell immunity to the conserved internal proteins of the virus. Despite this, LAIV has shown limited efficacy in recent years. One possible reason is that the conserved, internal genes of all current LAIVs derive from virus strains that were isolated between 1957 and 1960 and that, as a result, do not resemble currently circulating influenza viruses. We have therefore developed and tested a new LAIV, based on a currently circulating pandemic strain of influenza. Our results show that this new LAIV elicits improved protective immunity compared to a more conventional LAIV.

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Relative Effectiveness of the Cell-Cultured Quadrivalent Influenza Vaccine Compared to Standard, Egg-derived Quadrivalent Influenza Vaccines in Preventing Influenza-like Illness in 2017–2018

Clinical Infectious Diseases ◽

10.1093/cid/ciaa371 ◽

2020 ◽

Vol 71 (10) ◽

pp. e665-e671 ◽

Cited By ~ 4

Author(s):

Constantina Boikos ◽

Gregg C Sylvester ◽

John S Sampalis ◽

James A Mansi

Keyword(s):

Cell Culture ◽

Health Status ◽

Influenza Vaccine ◽

Influenza Season ◽

Relative Effectiveness ◽

Geographic Region ◽

Influenza Vaccines ◽

Study Cohort ◽

Influenza Like Illness ◽

Adaptive Mutations

Abstract Background Influenza antigens may undergo adaptive mutations during egg-based vaccine production. In the 2017–2018 influenza season, quadrivalent, inactivated cell-derived influenza vaccine (ccIIV4) vaccine was produced using A(H3N2) seed virus propagated exclusively in cell culture, thus lacking egg adaptive changes. This United States study estimated relative vaccine effectiveness (rVE) of ccIIV4 vs egg-derived quadrivalent vaccines (egg-derived IIV4) for that season. Methods Vaccination, outcome, and covariate data were ascertained retrospectively from a electronic medical record (EMR) dataset and analyzed. The study cohort included patients ≥ 4 years of age. rVE was estimated against influenza-like illness (ILI) using diagnostic International Classification of Diseases, Ninth or Tenth Revision codes. The adjusted odds ratios used to derive rVE estimates were estimated from multivariable logistic regression models adjusted for age, sex, race/ethnicity, geographic region, and health status. Results Overall, 92 187 individuals had a primary care EMR record of ccIIV4 and 1 261 675 had a record of egg-derived IIV4. In the ccIIV4 group, 1705 narrowly defined ILI events occurred, and 25 645 occurred in the standard egg-derived IIV4 group. Crude rVE was 9.2% (95% confidence interval [CI], 4.6%–13.6%). When adjusted for age, sex, health status, comorbidities, and geographic region, the estimated rVE changed to 36.2% (95% CI, 26.1%–44.9%). Conclusions ccIIV4, derived from A(H3N2) seed virus propagated exclusively in cell culture, was more effective than egg-derived IIV4 in preventing ILI during the 2017–2018 influenza season. This result suggests that cell-derived influenza vaccines may have greater effectiveness than standard egg-derived vaccines.

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Live attenuated influenza vaccine strains elicit a greater innate immune response than antigenically-matched seasonal influenza viruses during infection of human nasal epithelial cell cultures

Vaccine ◽

10.1016/j.vaccine.2013.12.069 ◽

2014 ◽

Vol 32 (15) ◽

pp. 1761-1767 ◽

Cited By ~ 19

Author(s):

William A. Fischer II ◽

Kelly D. Chason ◽

Missy Brighton ◽

Ilona Jaspers

Keyword(s):

Immune Response ◽

Epithelial Cell ◽

Influenza Vaccine ◽

Cell Cultures ◽

Seasonal Influenza ◽

Influenza Viruses ◽

Innate Immune ◽

Nasal Epithelial Cell ◽

Human Nasal Epithelial Cell ◽

Live Attenuated Influenza Vaccine

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Sequential delivery of LAIV and SARS-CoV-2 in the ferret model can reduce SARS-CoV-2 shedding and does not result in enhanced lung pathology

10.1101/2021.02.01.429110 ◽

2021 ◽

Author(s):

Kathryn A. Ryan ◽

Katarzyna E. Schewe ◽

Jonathan Crowe ◽

Susan A. Fotheringham ◽

Yper Hall ◽

...

Keyword(s):

Influenza Vaccine ◽

Severe Disease ◽

Influenza Viruses ◽

Lung Pathology ◽

Upper Respiratory Tract ◽

Mild Disease ◽

Live Attenuated Influenza Vaccine ◽

Disease Outcomes ◽

Close Proximity ◽

Upper Respiratory

AbstractCo-circulation of SARS-CoV-2 and influenza viruses could pose unpredictable risks to health systems globally, with recent studies suggesting more severe disease outcomes in co-infected patients. The lack of a readily available COVID-19 vaccine has reinforced the importance of influenza vaccine programmes during the COVID-19 pandemic. Live Attenuated Influenza Vaccine (LAIV) is an important tool in protecting against influenza, particularly in children. However, it is unknown whether LAIV administration might influence the outcomes of acute SARS-CoV-2 infection or disease. To investigate this, quadrivalent LAIV (QLAIV) was administered to ferrets 3 days pre- or post-SARS-CoV-2 infection. LAIV administration did not exacerbate SARS-CoV-2 disease course or lung pathology with either regimen. Additionally, LAIV administered prior to SARS-CoV-2 infection significantly reduced SARS-CoV-2 replication and shedding in the upper respiratory tract (URT). We conclude that LAIV administration in close proximity to SARS-CoV-2 infection does not exacerbate mild disease and can reduce SARS-CoV-2 shedding.

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A Mutated PB1 Residue 319 Synergizes with the PB2 N265S Mutation of the Live Attenuated Influenza Vaccine to Convey Temperature Sensitivity

Viruses ◽

10.3390/v12111246 ◽

2020 ◽

Vol 12 (11) ◽

pp. 1246

Author(s):

Andrew Cox ◽

Jordana Schmierer ◽

Josephine D’Angelo ◽

Andrew Smith ◽

Dustyn Levenson ◽

...

Keyword(s):

Temperature Sensitivity ◽

Genetic Background ◽

Influenza A ◽

Polymerase Activity ◽

Influenza Vaccines ◽

Potential Utility ◽

Live Vaccines ◽

Live Attenuated Influenza Vaccine ◽

Complete Set ◽

New Generation

Current influenza vaccines have modest efficacy. This is especially true for current live attenuated influenza vaccines (LAIV), which have been inferior to the inactivated versions in recent years. Therefore, a new generation of live vaccines may be needed. We previously showed that a mutation at PB1 residue 319 confers enhanced temperature sensitivity and attenuation in an LAIV constructed in the genetic background of the mouse-adapted Influenza A Virus (IAV) strain A/PR/8/34 (PR8). Here, we describe the origin/discovery of this unique mutation and demonstrate that, when combined with the PB2 N265S mutation of LAIV, it conveys an even greater level of temperature sensitivity and attenuation on PR8 than the complete set of attenuating mutations from LAIV. Furthermore, we show that the combined PB1 L319Q and PB2 N265S mutations confer temperature sensitivity on IAV polymerase activity in two different genetic backgrounds, PR8 and A/Cal/04/09. Collectively, these findings show that the PB2 LAIV mutation synergizes with a mutation in PB1 and may have potential utility for improving LAIVs.

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Effectiveness of 2 Influenza Vaccines in Nationwide Cohorts of Finnish 2-Year-Old Children in the Seasons 2015–2016 Through 2017–2018

Clinical Infectious Diseases ◽

10.1093/cid/ciaa050 ◽

2020 ◽

Vol 71 (8) ◽

pp. e255-e261

Author(s):

Ulrike Baum ◽

Sangita Kulathinal ◽

Kari Auranen ◽

Hanna Nohynek

Keyword(s):

Influenza Vaccine ◽

Virus Type ◽

Real Life ◽

Vaccine Effectiveness ◽

Influenza Vaccines ◽

Type B ◽

Live Attenuated Influenza Vaccine ◽

Vaccine Type ◽

Decision Making Processes ◽

Study Population

Abstract Background From 2015–2016 through 2017–2018, injectable, trivalent inactivated influenza vaccines (IIV3) and a nasal spray, tetravalent live-attenuated influenza vaccine (LAIV4) were used in parallel in Finland. To understand how well vaccination with each vaccine type protected children against influenza under real-life conditions, vaccine effectiveness in 2-year-olds was estimated for all 3 seasons. Methods Each season, a nationwide register-based cohort study was conducted. The study population comprised 60 088, 60 860, and 60 345 children in 2015–2016, 2016–2017, and 2017–2018, respectively. Laboratory-confirmed influenza was the study outcome. Seasonal influenza vaccination with either LAIV4 or IIV3 was the time-dependent exposure of interest. Vaccine effectiveness was defined as 1 minus the hazard ratio comparing vaccinated with unvaccinated children. Results From 2015–2016 through 2017–2018, the effectiveness of LAIV4 against influenza of any virus type was estimated at 54.2% (95% confidence interval, 32.2–69.0%), 20.3% (−12.7%, 43.6%), and 30.5% (10.9–45.9%); the corresponding effectiveness of IIV3 was 77.2% (48.9–89.8%), 24.5% (−29.8%, 56.1%), and −20.1% (−61.5%, 10.7%). Neither influenza vaccine clearly excelled in protecting children. The LAIV4 effectiveness against type B was greater than against type A and greater than the IIV3 effectiveness against type B. Conclusions To understand how influenza vaccines could be improved, vaccine effectiveness must be analyzed by vaccine and virus type. Effectiveness estimates also expressing overall protection levels are needed to guide individual and programmatic decision-making processes. Supported by this analysis, the vaccination program in Finland now recommends LAIV4 and injectable, tetravalent inactivated influenza vaccines replacing IIV3.

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Comparisons of the Humoral and Cellular Immune Responses Induced by Live Attenuated Influenza Vaccine and Inactivated Influenza Vaccine in Adults

Clinical and Vaccine Immunology ◽

10.1128/cvi.00414-16 ◽

2016 ◽

Vol 24 (1) ◽

Cited By ~ 42

Author(s):

Daniel F. Hoft ◽

Kathleen R. Lottenbach ◽

Azra Blazevic ◽

Aldin Turan ◽

Tamara P. Blevins ◽

...

Keyword(s):

Influenza Virus ◽

T Cell ◽

Young Children ◽

Influenza Vaccine ◽

T Cell Responses ◽

Influenza Vaccines ◽

Secretory Iga ◽

Cell Responses ◽

Live Attenuated Influenza Vaccine ◽

Cellular Immune

ABSTRACT Both live attenuated influenza vaccines (LAIV) and inactivated influenza vaccines (IIV) induce protective immunity against influenza. There is evidence that LAIV induces superior protection in children, whereas IIV may induce superior protection in adults. The immune mechanisms responsible for these differences have not been identified. We previously compared LAIV and IIV in young children of 6 to 36 months of age, and we demonstrated that while both induced similar hemagglutination inhibition (HAI) antibody responses, only LAIV induced significant increases in T cell responses. In the present study, 37 healthy adult subjects of 18 to 49 years of age were randomized to receive seasonal influenza vaccination with LAIV or IIV. Influenza virus-specific HAI, T cell, and secretory IgA (sIgA) responses were studied pre- and postvaccination. In contrast to the responses seen in young children, LAIV induced only minimal increases in serum HAI responses in adults, which were significantly lower than the responses induced by IIV. Both LAIV and IIV similarly induced only transient T cell responses to replication-competent whole virus in adults. In contrast, influenza virus-specific sIgA responses were induced more strongly by LAIV than by IIV. Our previous studies suggest that LAIV may be more protective than IIV in young children not previously exposed to influenza virus or influenza vaccines due to increased vaccine-induced T cell and/or sIgA responses. Our current work suggests that in adults with extensive and partially cross-reactive preexisting influenza immunity, LAIV boosting of sIgA responses to hemagglutinin (HA) and non-HA antigenic targets expressed by circulating influenza virus strains may be an important additional mechanism of vaccine-induced immunity.

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Universal Live-Attenuated Influenza Vaccine Candidates Expressing Multiple M2e Epitopes Protect Ferrets against a High-Dose Heterologous Virus Challenge

Viruses ◽

10.3390/v13071280 ◽

2021 ◽

Vol 13 (7) ◽

pp. 1280

Author(s):

Daria Mezhenskaya ◽

Irina Isakova-Sivak ◽

Victoria Matyushenko ◽

Svetlana Donina ◽

Andrey Rekstin ◽

...

Keyword(s):

Influenza Virus ◽

Influenza Vaccine ◽

Swine Influenza ◽

Influenza Viruses ◽

High Dose ◽

Virus Challenge ◽

Consensus Sequences ◽

Degree Of Protection ◽

Live Attenuated Influenza Vaccine ◽

Universal Influenza Vaccine

The development of an influenza vaccine with broad protection and durability remains an attractive idea due to the high mutation rate of the influenza virus. An extracellular domain of Matrix 2 protein (M2e) is among the most attractive target for the universal influenza vaccine owing to its high conservancy rate. Here, we generated two recombinant live attenuated influenza vaccine (LAIV) candidates encoding four M2e epitopes representing consensus sequences of human, avian and swine influenza viruses, and studied them in a preclinical ferret model. Both LAIV+4M2e viruses induced higher levels of M2e-specific antibodies compared to the control LAIV strain, with the LAIV/HA+4M2e candidate being significantly more immunogenic than the LAIV/NS+4M2e counterpart. A high-dose heterosubtypic influenza virus challenge revealed the highest degree of protection after immunization with LAIV/HA+4M2e strain, followed by the NS-modified LAIV and the classical LAIV virus. Furthermore, only the immune sera from the LAIV/HA+4M2e-immunized ferrets protected mice from a panel of lethal influenza viruses encoding M genes of various origins. These data suggest that the improved cross-protection of the LAIV/HA+4M2e universal influenza vaccine candidate was mediated by the M2e-targeted antibodies. Taking into account the safety profile and improved cross-protective potential, the LAIV/HA+4M2e vaccine warrants its further evaluation in a phase I clinical trial.

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Safety, Immunogenicity, and Protective Efficacy of a Chimeric A/B Live Attenuated Influenza Vaccine in a Mouse Model

Microorganisms ◽

10.3390/microorganisms9020259 ◽

2021 ◽

Vol 9 (2) ◽

pp. 259

Author(s):

Ekaterina Stepanova ◽

Elena Krutikova ◽

Pei-Fong Wong ◽

Victoria Matyushenko ◽

Ekaterina Bazhenova ◽

...

Keyword(s):

T Cell ◽

Influenza Vaccine ◽

Influenza A ◽

Type A ◽

Influenza Viruses ◽

Effector Memory ◽

Influenza B Virus ◽

Influenza B ◽

Live Attenuated Influenza Vaccine ◽

B Virus

Influenza A and B viruses cause significant morbidity and mortality worldwide. Current influenza vaccines are composed of three or four strains: A/H1N1, A/H3N2, and B (Victoria and Yamagata lineages). It is of great interest if immunization against both type A and B influenza viruses can be combined in a single vaccine strain, thus reducing the cost of vaccine production and the possibility of strain interference within the multicomponent vaccine. In the current study, we developed an experimental live cold-adapted influenza intertype reassortant (influenza A and B) vaccine on the live attenuated influenza vaccine (LAIV) A/Leningrad/134/17/57 backbone. Hemagglutinin (HA) and neuraminidase (NA) functional domains were inherited from the influenza B/Brisbane/60/2008 strain, whereas their packaging signals were substituted with appropriate fragments of influenza A virus genes. The recombinant A/B virus efficiently replicated in eggs and Madin–Darby Canine Kidney (MDCK) cells under optimal conditions, temperature-sensitive phenotype was maintained, and its antigenic properties matched the influenza B parental virus. The chimeric vaccine was attenuated in mice: after intranasal immunization, viral replication was seen only in nasal turbinates but not in the lungs. Immunological studies demonstrated the induction of IgG antibody responses against the influenza A and B virus, whereas hemagglutination inhibition (HAI) and neutralizing antibodies were detected only against the influenza B virus, resulting in significant protection of immunized animals against influenza B virus challenge. IFNγ-secreting CD8 effector memory T cells (CD44+CD62L−) were detected in mouse splenocytes after stimulation with the specific influenza A peptide (NP366); however, the T-cell response was not sufficient to protect animals against infection with a high-dose mouse-adapted A/California/07/2009 (H1N1pdm09) virus, most probably due to the mismatch of key T-cell epitopes of the H1N1 virus and the LAIV backbone. Overall, generation of the chimeric A/B LAIV virus on a licensed LAIV backbone demonstrated prospects for the development of safe and efficacious vaccine candidates that afford combined protection against both type A and type B influenza viruses; however, further optimization of the T-cell epitope content within the LAIV backbone may be required.

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