Use of lentiviral pseudotypes as an alternative to reassortant or Triton X-100-treated wild-type Influenza viruses in the neuraminidase inhibition enzyme-linked lectin assay

AbstractWhile vaccines remain the best tool for preventing influenza virus infections, they have demonstrated low to moderate effectiveness in recent years. Seasonal influenza vaccines typically consist of wild-type influenza A and B viruses that are limited in their ability to elicit protective immune responses against co-circulating influenza virus variant strains. Improved influenza virus vaccines need to elicit protective immune responses against multiple influenza virus drift variants within each season. Broadly reactive vaccine candidates potentially provide a solution to this problem, but their efficacy may begin to wane as influenza viruses naturally mutate through processes that mediates drift. Thus, it is necessary to develop a method that commercial vaccine manufacturers can use to update broadly reactive vaccine antigens to better protect against future and currently circulating viral variants. Building upon the COBRA technology, nine next-generation H3N2 influenza hemagglutinin (HA) vaccines were designed using a next generation algorithm and design methodology. These next-generation broadly reactive COBRA H3 HA vaccines were superior to wild-type HA vaccines at eliciting antibodies with high HAI activity against a panel of historical and co-circulating H3N2 influenza viruses isolated over the last 15 years, as well as the ability to neutralize future emerging H3N2 isolates.

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Next Generation of Computationally Optimized Broadly Reactive HA Vaccines Elicited Cross-Reactive Immune Responses and Provided Protection against H1N1 Virus Infection

Vaccines ◽

10.3390/vaccines9070793 ◽

2021 ◽

Vol 9 (7) ◽

pp. 793

Author(s):

Ying Huang ◽

Monique S. França ◽

James D. Allen ◽

Hua Shi ◽

Ted M. Ross

Keyword(s):

Influenza Virus ◽

Virus Infection ◽

Immune Responses ◽

H1n1 Virus ◽

Influenza Virus Infection ◽

H1n1 Influenza ◽

Influenza Viruses ◽

Next Generation ◽

Wild Type ◽

Influenza A H1n1

Vaccination is the best way to prevent influenza virus infections, but the diversity of antigenically distinct isolates is a persistent challenge for vaccine development. In order to conquer the antigenic variability and improve influenza virus vaccine efficacy, our research group has developed computationally optimized broadly reactive antigens (COBRAs) in the form of recombinant hemagglutinins (rHAs) to elicit broader immune responses. However, previous COBRA H1N1 vaccines do not elicit immune responses that neutralize H1N1 virus strains in circulation during the recent years. In order to update our COBRA vaccine, two new candidate COBRA HA vaccines, Y2 and Y4, were generated using a new seasonal-based COBRA methodology derived from H1N1 isolates that circulated during 2013–2019. In this study, the effectiveness of COBRA Y2 and Y4 vaccines were evaluated in mice, and the elicited immune responses were compared to those generated by historical H1 COBRA HA and wild-type H1N1 HA vaccines. Mice vaccinated with the next generation COBRA HA vaccines effectively protected against morbidity and mortality after infection with H1N1 influenza viruses. The antibodies elicited by the COBRA HA vaccines were highly cross-reactive with influenza A (H1N1) pdm09-like viruses isolated from 2009 to 2021, especially with the most recent circulating viruses from 2019 to 2021. Furthermore, viral loads in lungs of mice vaccinated with Y2 and Y4 were dramatically reduced to low or undetectable levels, resulting in minimal lung injury compared to wild-type HA vaccines following H1N1 influenza virus infection.

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Evaluation of Replication and Cross-Reactive Antibody Responses of H2 Subtype Influenza Viruses in Mice and Ferrets

Journal of Virology ◽

10.1128/jvi.00511-10 ◽

2010 ◽

Vol 84 (15) ◽

pp. 7695-7702 ◽

Cited By ~ 37

Author(s):

Grace L. Chen ◽

Elaine W. Lamirande ◽

Chin-Fen Yang ◽

Hong Jin ◽

George Kemble ◽

...

Keyword(s):

Influenza Virus ◽

Pandemic Influenza ◽

Vaccine Candidate ◽

Influenza Virus Vaccine ◽

Influenza Viruses ◽

Antibody Responses ◽

Wild Type ◽

Preparedness Planning ◽

Kinetics Of ◽

Reactive Antibody

ABSTRACT H2 influenza viruses have not circulated in humans since 1968, and therefore a large segment of the population would likely be susceptible to infection should H2 influenza viruses reemerge. The development of an H2 pandemic influenza virus vaccine candidate should therefore be considered a priority in pandemic influenza preparedness planning. We selected a group of geographically and temporally diverse wild-type H2 influenza viruses and evaluated the kinetics of replication and compared the ability of these viruses to induce a broadly cross-reactive antibody response in mice and ferrets. In both mice and ferrets, A/Japan/305/1957 (H2N2), A/mallard/NY/1978 (H2N2), and A/swine/MO/2006 (H2N3) elicited the broadest cross-reactive antibody responses against heterologous H2 influenza viruses as measured by hemagglutination inhibition and microneutralization assays. These data suggested that these three viruses may be suitable candidates for development as live attenuated H2 pandemic influenza virus vaccines.

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Broadly Reactive H2 Hemagglutinin Vaccines Elicit Cross-Reactive Antibodies in Ferrets Preimmune to Seasonal Influenza A Viruses

mSphere ◽

10.1128/msphere.00052-21 ◽

2021 ◽

Vol 6 (2) ◽

Author(s):

Z. Beau Reneer ◽

Amanda L. Skarlupka ◽

Parker J. Jamieson ◽

Ted M. Ross

Keyword(s):

Influenza Virus ◽

Immune Responses ◽

Recombinant Proteins ◽

Human Population ◽

Influenza A ◽

Seasonal Influenza ◽

Influenza Viruses ◽

Wild Type ◽

Influenza A Viruses ◽

Global Pandemic

ABSTRACT Influenza vaccines have traditionally been tested in naive mice and ferrets. However, humans are first exposed to influenza viruses within the first few years of their lives. Therefore, there is a pressing need to test influenza virus vaccines in animal models that have been previously exposed to influenza viruses before being vaccinated. In this study, previously described H2 computationally optimized broadly reactive antigen (COBRA) hemagglutinin (HA) vaccines (Z1 and Z5) were tested in influenza virus “preimmune” ferret models. Ferrets were infected with historical, seasonal influenza viruses to establish preimmunity. These preimmune ferrets were then vaccinated with either COBRA H2 HA recombinant proteins or wild-type H2 HA recombinant proteins in a prime-boost regimen. A set of naive preimmune or nonpreimmune ferrets were also vaccinated to control for the effects of the multiple different preimmunities. All of the ferrets were then challenged with a swine H2N3 influenza virus. Ferrets with preexisting immune responses influenced recombinant H2 HA-elicited antibodies following vaccination, as measured by hemagglutination inhibition (HAI) and classical neutralization assays. Having both H3N2 and H1N1 immunological memory regardless of the order of exposure significantly decreased viral nasal wash titers and completely protected all ferrets from both morbidity and mortality, including the mock-vaccinated ferrets in the group. While the vast majority of the preimmune ferrets were protected from both morbidity and mortality across all of the different preimmunities, the Z1 COBRA HA-vaccinated ferrets had significantly higher antibody titers and recognized the highest number of H2 influenza viruses in a classical neutralization assay compared to the other H2 HA vaccines. IMPORTANCE H1N1 and H3N2 influenza viruses have cocirculated in the human population since 1977. Nearly every human alive today has antibodies and memory B and T cells against these two subtypes of influenza viruses. H2N2 influenza viruses caused the 1957 global pandemic and people born after 1968 have never been exposed to H2 influenza viruses. It is quite likely that a future H2 influenza virus could transmit within the human population and start a new global pandemic, since the majority of people alive today are immunologically naive to viruses of this subtype. Therefore, an effective vaccine for H2 influenza viruses should be tested in an animal model with previous exposure to influenza viruses that have circulated in humans. Ferrets were infected with historical influenza A viruses to more accurately mimic the immune responses in people who have preexisting immune responses to seasonal influenza viruses. In this study, preimmune ferrets were vaccinated with wild-type (WT) and COBRA H2 recombinant HA proteins in order to examine the effects that preexisting immunity to seasonal human influenza viruses have on the elicitation of broadly cross-reactive antibodies from heterologous vaccination.

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A Host-Restricted Self-Attenuated Influenza Virus Provides Broad Pan-Influenza A Protection in a Mouse Model

Frontiers in Immunology ◽

10.3389/fimmu.2021.779223 ◽

2021 ◽

Vol 12 ◽

Author(s):

Minjin Kim ◽

Yucheol Cheong ◽

Jinhee Lee ◽

Jongkwan Lim ◽

Sanguine Byun ◽

...

Keyword(s):

Influenza Virus ◽

Mouse Model ◽

Neutralizing Antibodies ◽

Influenza A ◽

Protective Efficacy ◽

Influenza Viruses ◽

Cross Protection ◽

Infection Model ◽

Wild Type ◽

Group 1

Influenza virus infections can cause a broad range of symptoms, form mild respiratory problems to severe and fatal complications. While influenza virus poses a global health threat, the frequent antigenic change often significantly compromises the protective efficacy of seasonal vaccines, further increasing the vulnerability to viral infection. Therefore, it is in great need to employ strategies for the development of universal influenza vaccines (UIVs) which can elicit broad protection against diverse influenza viruses. Using a mouse infection model, we examined the breadth of protection of the caspase-triggered live attenuated influenza vaccine (ctLAIV), which was self-attenuated by the host caspase-dependent cleavage of internal viral proteins. A single vaccination in mice induced a broad reactive antibody response against four different influenza viruses, H1 and rH5 (HA group 1) and H3 and rH7 subtypes (HA group 2). Notably, despite the lack of detectable neutralizing antibodies, the vaccination provided heterosubtypic protection against the lethal challenge with the viruses. Sterile protection was confirmed by the complete absence of viral titers in the lungs and nasal turbinates after the challenge. Antibody-dependent cellular cytotoxicity (ADCC) activities of non-neutralizing antibodies contributed to cross-protection. The cross-protection remained robust even after in vivo depletion of T cells or NK cells, reflecting the strength and breadth of the antibody-dependent effector function. The robust mucosal secretion of sIgA reflects an additional level of cross-protection. Our data show that the host-restricted designer vaccine serves an option for developing a UIV, providing pan-influenza A protection against both group 1 and 2 influenza viruses. The present results of potency and breadth of protection from wild type and reassortant viruses addressed in the mouse model by single immunization merits further confirmation and validation, preferably in clinically relevant ferret models with wild type challenges.

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Application of a Biologically Contained Reporter System To Study Gain-of-Function H5N1 Influenza A Viruses with Pandemic Potential

mSphere ◽

10.1128/msphere.00423-20 ◽

2020 ◽

Vol 5 (4) ◽

Author(s):

Eva E. Spieler ◽

Eva Moritz ◽

Silke Stertz ◽

Benjamin G. Hale

Keyword(s):

Viral Genome ◽

Influenza A ◽

Risk Mitigation ◽

Genetically Modified ◽

Influenza Viruses ◽

Mitigation Strategies ◽

Wild Type ◽

Gain Of Function ◽

Ha Gene ◽

Risk Mitigation Strategies

ABSTRACT Natural adaptation of an antigenically novel avian influenza A virus (IAV) to be transmitted efficiently in humans has the potential to trigger a devastating pandemic. Understanding viral genetic determinants underlying adaptation is therefore critical for pandemic preparedness, as the knowledge gained enhances surveillance and eradication efforts, prepandemic vaccine design, and efficacy assessment of antivirals. However, this work has risks, as making gain-of-function substitutions in fully infectious IAVs may create a pathogen with pandemic potential. Thus, such experiments must be tightly controlled through physical and biological risk mitigation strategies. Here, we applied a previously described biological containment system for IAVs to a 2009 pandemic H1N1 strain and a highly pathogenic H5N1 strain. The system relies on deletion of the essential viral hemagglutinin (HA) gene, which is instead provided in trans, thereby restricting multicycle virus replication to genetically modified HA-complementing cells. In place of HA, a Renilla luciferase gene is inserted within the viral genome, and a live-cell luciferase substrate allows real-time quantitative monitoring of viral replication kinetics with a high dynamic range. We demonstrate that biologically contained IAV-like particles exhibit wild-type sensitivities to approved antivirals, including oseltamivir, zanamivir, and baloxavir. Furthermore, the inability of these IAV-like particles to genetically acquire the host-encoded HA allowed us to introduce gain-of-function substitutions in the H5 HA gene that promote mammalian transmissibility. Biologically contained “transmissible” H5N1 IAV-like particles exhibited wild-type sensitivities to approved antivirals, to the fusion inhibitor S20, and to neutralization by existing H5 monoclonal and polyclonal sera. This work represents a proof of principle that biologically contained IAV systems can be used to safely conduct selected gain-of-function experiments. IMPORTANCE Understanding how animal influenza viruses can adapt to spread in humans is critical to prepare for, and prevent, new pandemics. However, working safely with pathogens that have pandemic potential requires tight regulation and the use of high-level physical and biological risk mitigation strategies to stop accidental loss of containment. Here, we used a biological containment system for influenza viruses to study strains with pandemic potential. The system relies on deletion of the essential HA gene from the viral genome and its provision by a genetically modified cell line, to which virus propagation is therefore restricted. We show that this method permits safe handling of these pathogens, including gain-of-function variants, without the risk of generating fully infectious viruses. Furthermore, we demonstrate that this system can be used to assess virus sensitivity to both approved and experimental drugs, as well as the antigenic profile of viruses, important considerations for evaluating prepandemic vaccine and antiviral strategies.

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PB1-mediated virulence attenuation of H5N1 influenza virus in mice is associated with PB2

Journal of General Virology ◽

10.1099/vir.0.030718-0 ◽

2011 ◽

Vol 92 (6) ◽

pp. 1435-1444 ◽

Cited By ~ 20

Author(s):

Jing Li ◽

Yongqiang Li ◽

Yi Hu ◽

Guohui Chang ◽

Wei Sun ◽

...

Keyword(s):

Influenza Virus ◽

Recombinant Virus ◽

Influenza Viruses ◽

H5n1 Influenza Virus ◽

Wild Type ◽

Small Plaque ◽

Large Plaque ◽

H5n1 Influenza ◽

Virulence Attenuation ◽

N Terminus

H5N1 avian influenza viruses demonstrate different phenotypes, such as pathogenicity after one or serial passages in mammalian hosts or cells. To establish the molecular basis of these phenotypes, we cloned isolates from the lungs of mice infected with human A/Vietnam/1194/2004 (H5N1) influenza virus. Large-plaque isolates were less pathogenic to mice than small-plaque isolates. Genome sequencing revealed that the small-plaque and large-plaque isolates differed in several amino acids. In order to assess their effects on pathogenicity in mice, two amino acid changes common to attenuated isolates, one in PB2 (I63T) and the other in PB1 (T677M), were inserted into a wild-type recombinant virus construct. The PB2 (I63T) or PB1 (T677M) mutations alone did not alter the phenotype of H5N1 virus, whereas recombinant virus with both mutations was less pathogenic than the wild-type recombinant virus. Furthermore, the PB1 (T677M) mutation showed a lower replication efficiency, although it had higher polymerase activity. The recombinant virus with the PB2 (63T) mutation replicated as well as the wild-type recombinant virus. These results suggest that the C terminus of PB1 of H5N1 influenza virus mediates virulence attenuation of H5N1 influenza virus in mice, associating with the N terminus of PB2. However, the role of the N terminus of PB2 in virulence attenuation in mice remains unclear.

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Mutations in the NS1 Protein of Swine Influenza Virus Impair Anti-Interferon Activity and Confer Attenuation in Pigs

Journal of Virology ◽

10.1128/jvi.79.12.7535-7543.2005 ◽

2005 ◽

Vol 79 (12) ◽

pp. 7535-7543 ◽

Cited By ~ 176

Author(s):

Alicia Solórzano ◽

Richard J. Webby ◽

Kelly M. Lager ◽

Bruce H. Janke ◽

Adolfo García-Sastre ◽

...

Keyword(s):

Influenza Virus ◽

Nonstructural Protein ◽

Swine Influenza Virus ◽

Swine Influenza ◽

Influenza Viruses ◽

Model Systems ◽

Ns1 Protein ◽

Wild Type ◽

Virulence Attenuation

ABSTRACT It has been shown previously that the nonstructural protein NS1 of influenza virus is an alpha/beta interferon (IFN-α/β) antagonist, both in vitro and in experimental animal model systems. However, evidence of this function in a natural host has not yet been obtained. Here we investigated the role of the NS1 protein in the virulence of a swine influenza virus (SIV) isolate in pigs by using reverse genetics. The virulent wild-type A/Swine/Texas/4199-2/98 (TX/98) virus and various mutants encoding carboxy-truncated NS1 proteins were rescued. Growth properties of TX/98 viruses with mutated NS1, induction of IFN in tissue culture, and virulence-attenuation in pigs were analyzed and compared to those of the recombinant wild-type TX/98 virus. Our results indicate that deletions in the NS1 protein decrease the ability of the TX/98 virus to prevent IFN-α/β synthesis in pig cells. Moreover, all NS1 mutant viruses were attenuated in pigs, and this correlated with the amount of IFN-α/β induced in vitro. These data suggest that the NS1 protein of SIV is a virulence factor. Due to their attenuation, NS1-mutated swine influenza viruses might have a great potential as live attenuated vaccine candidates against SIV infections of pigs.

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Computationally Optimized Broadly Reactive H2 HA Influenza Vaccines Elicited Broadly Cross-Reactive Antibodies and Protected Mice from Viral Challenges

Journal of Virology ◽

10.1128/jvi.01526-20 ◽

2020 ◽

Vol 95 (2) ◽

pp. e01526-20

Author(s):

Z. Beau Reneer ◽

Parker J. Jamieson ◽

Amanda L. Skarlupka ◽

Ying Huang ◽

Ted M. Ross

Keyword(s):

Influenza Virus ◽

Influenza Vaccine ◽

Neutralizing Antibodies ◽

Influenza Pandemic ◽

Mammalian Species ◽

Influenza Viruses ◽

Wild Type Virus ◽

Wild Type ◽

The 1950S ◽

Future Work

ABSTRACTInfluenza viruses have caused numerous pandemics throughout human history. The 1957 influenza pandemic was initiated by an H2N2 influenza virus. This H2N2 influenza virus was the result of a reassortment event between a circulating H2N2 avian virus and the seasonal H1N1 viruses in humans. Previously, our group has demonstrated the effectiveness of hemagglutinin (HA) antigens derived using computationally optimized broadly reactive antigen (COBRA) methodology against H1N1, H3N2, and H5N1 viruses. Using the COBRA methodology, H2 HA COBRA antigens were designed using sequences from H2N2 viruses isolated from humans in the 1950s and 1960s, as well as H2Nx viruses isolated from avian and mammalian species between the 1950s and 2016. In this study, the effectiveness of H2 COBRA HA antigens (Z1, Z3, Z5, and Z7) was evaluated in DBA/2J mice and compared to that of wild-type H2 HA antigens. The COBRA HA vaccines elicited neutralizing antibodies to the majority of viruses in our H2 HA panel and across all three clades as measured by hemagglutination inhibition (HAI) and neutralization assays. Comparatively, several wild-type HA vaccines elicited antibodies against a majority of the viruses in the H2 HA panel. DBA/2J mice vaccinated with COBRA vaccines showed increase survival for all three viral challenges compared to the wild-type H2 vaccines. In particular, the Z1 COBRA is a promising candidate for future work toward a pandemic H2 influenza vaccine.IMPORTANCE H2N2 influenza has caused at least one pandemic in the past. Given that individuals born after 1968 have not been exposed to H2N2 influenza viruses, a future pandemic caused by H2 influenza is likely. An effective H2 influenza vaccine would need to elicit broadly cross-reactive antibodies to multiple H2 influenza viruses. Choosing a wild-type virus to create a vaccine may elicit a narrow immune response and not protect against multiple H2 influenza viruses. COBRA H2 HA vaccines were developed and evaluated in mice along with wild-type H2 HA vaccines. Multiple COBRA H2 HA vaccines protected mice from all three viral challenges and produced broadly cross-reactive neutralizing antibodies to H2 influenza viruses.

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