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

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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Next generation methodology for updating HA vaccines against emerging human seasonal influenza A(H3N2) viruses

Scientific Reports ◽

10.1038/s41598-020-79590-7 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

James D. Allen ◽

Ted M. Ross

Keyword(s):

Influenza Virus ◽

Immune Responses ◽

Influenza A ◽

Seasonal Influenza ◽

Influenza Viruses ◽

Next Generation ◽

Virus Infections ◽

Wild Type ◽

Influenza Hemagglutinin ◽

H3n2 Influenza

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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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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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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Broadly Neutralizing Anti-Influenza Virus Antibodies: Enhancement of Neutralizing Potency in Polyclonal Mixtures and IgA Backbones

Journal of Virology ◽

10.1128/jvi.03099-14 ◽

2015 ◽

Vol 89 (7) ◽

pp. 3610-3618 ◽

Cited By ~ 50

Author(s):

Wenqian He ◽

Caitlin E. Mullarkey ◽

J. Andrew Duty ◽

Thomas M. Moran ◽

Peter Palese ◽

...

Keyword(s):

Influenza Virus ◽

Monoclonal Antibodies ◽

Virus Vaccine ◽

Neutralizing Antibodies ◽

Influenza A ◽

Human Peripheral Blood ◽

Influenza Virus Vaccine ◽

Influenza Viruses ◽

Broadly Neutralizing Antibodies ◽

Variable Regions

ABSTRACTCurrent influenza virus vaccines rely upon the accurate prediction of circulating virus strains months in advance of the actual influenza season in order to allow time for vaccine manufacture. Unfortunately, mismatches occur frequently, and even when perfect matches are achieved, suboptimal vaccine efficacy leaves several high-risk populations vulnerable to infection. However, the recent discovery of broadly neutralizing antibodies that target the hemagglutinin (HA) stalk domain has renewed hope that the development of “universal” influenza virus vaccines may be within reach. Here, we examine the functions of influenza A virus hemagglutinin stalk-binding antibodies in an endogenous setting, i.e., as polyclonal preparations isolated from human sera. Relative to monoclonal antibodies that bind to the HA head domain, the neutralization potency of monoclonal stalk-binding antibodies was vastly inferiorin vitrobut was enhanced by several orders of magnitude in the polyclonal context. Furthermore, we demonstrated a surprising enhancement in IgA-mediated HA stalk neutralization relative to that achieved by antibodies of IgG isotypes. Mechanistically, this could be explained in two ways. Identical variable regions consistently neutralized virus more potently when in an IgA backbone compared to an IgG backbone. In addition, HA-specific memory B cells isolated from human peripheral blood were more likely to be stalk specific when secreting antibodies of IgA isotypes compared to those secreting IgG. Taken together, our data provide strong evidence that HA stalk-binding antibodies perform optimally when in a polyclonal context and that the targeted elicitation of HA stalk-specific IgA should be an important consideration during “universal” influenza virus vaccine design.IMPORTANCEInfluenza viruses remain one of the most worrisome global public health threats due to their capacity to cause pandemics. While seasonal vaccines fail to protect against the emergence of pandemic strains, a new class of broadly neutralizing antibodies has been recently discovered and may be the key to developing a “universal” influenza virus vaccine. While much has been learned about the biology of these antibodies, most studies have focused only on monoclonal antibodies of IgG subtypes. However, the study of monoclonal antibodies often fails to capture the complexity of antibody functions that occur during natural polyclonal responses. Here, we provide the first detailed analyses of the biological activity of these antibodies in polyclonal contexts, comparing both IgG and IgA isotypes isolated from human donors. The striking differences observed in the functional properties of broadly neutralizing antibodies in polyclonal contexts will be essential for guiding design of “universal” influenza virus vaccines and therapeutics.

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A small-molecule fragment that emulates binding of receptor and broadly neutralizing antibodies to influenza A hemagglutinin

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1801999115 ◽

2018 ◽

Vol 115 (16) ◽

pp. 4240-4245 ◽

Cited By ~ 12

Author(s):

Rameshwar U. Kadam ◽

Ian A. Wilson

Keyword(s):

Influenza Virus ◽

Small Molecule ◽

Receptor Binding ◽

Neutralizing Antibodies ◽

Influenza A ◽

Binding Mode ◽

Host Cells ◽

Viral Pathogen ◽

Group 2 ◽

Group 1

The influenza virus hemagglutinin (HA) glycoprotein mediates receptor binding and membrane fusion during viral entry in host cells. Blocking these key steps in viral infection has applications for development of novel antiinfluenza therapeutics as well as vaccines. However, the lack of structural information on how small molecules can gain a foothold in the small, shallow receptor-binding site (RBS) has hindered drug design against this important target on the viral pathogen. Here, we report on the serendipitous crystallization-based discovery of a small-molecule N-cyclohexyltaurine, commonly known as the buffering agent CHES, that is able to bind to both group-1 and group-2 HAs of influenza A viruses. X-ray structural characterization of group-1 H5N1 A/Vietnam/1203/2004 (H5/Viet) and group-2 H3N2 A/Hong Kong/1/1968 (H3/HK68) HAs at 2.0-Å and 2.57-Å resolution, respectively, revealed that N-cyclohexyltaurine binds to the heart of the conserved HA RBS. N-cyclohexyltaurine mimics the binding mode of the natural receptor sialic acid and RBS-targeting bnAbs through formation of similar hydrogen bonds and CH-π interactions with the HA. In H3/HK68, N-cyclohexyltaurine also binds to a conserved pocket in the stem region, thereby exhibiting a dual-binding mode in group-2 HAs. These long-awaited structural insights into RBS recognition by a noncarbohydrate-based small molecule enhance our knowledge of how to target this important functional site and can serve as a template to guide the development of novel broad-spectrum small-molecule therapeutics against influenza virus.

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Comparative studies of infectivity, immunogenicity and cross-protective efficacy of live attenuated influenza vaccines containing nucleoprotein from cold-adapted or wild-type influenza virus in a mouse model

Virology ◽

10.1016/j.virol.2016.10.027 ◽

2017 ◽

Vol 500 ◽

pp. 209-217 ◽

Cited By ~ 15

Author(s):

Irina Isakova-Sivak ◽

Daniil Korenkov ◽

Tatiana Smolonogina ◽

Tatiana Tretiak ◽

Svetlana Donina ◽

...

Keyword(s):

Influenza Virus ◽

Mouse Model ◽

Comparative Studies ◽

Protective Efficacy ◽

Influenza Vaccines ◽

Wild Type ◽

Cold Adapted

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A Genetically Engineered Waterfowl Influenza Virus with a Deletion in the Stalk of the Neuraminidase Has Increased Virulence for Chickens

Journal of Virology ◽

10.1128/jvi.01581-09 ◽

2009 ◽

Vol 84 (2) ◽

pp. 940-952 ◽

Cited By ~ 95

Author(s):

S. Munier ◽

T. Larcher ◽

F. Cormier-Aline ◽

D. Soubieux ◽

B. Su ◽

...

Keyword(s):

Influenza Virus ◽

Influenza A ◽

Molecular Mechanisms ◽

Transforming Growth Factor ◽

Cultured Cells ◽

Genetically Engineered ◽

Influenza Viruses ◽

Wild Type Virus ◽

Wild Type ◽

Moderate Growth

ABSTRACT A deletion of about 20 amino acids in the stalk of the neuraminidase (NA) is frequently detected upon transmission of influenza A viruses from waterfowl to domestic poultry. Using reverse genetics, a recombinant virus derived from a wild duck influenza virus isolate, A/Mallard/Marquenterre/Z237/83 (MZ), and an NA stalk deletion variant (MZ-delNA) were produced. Compared to the wild type, the MZ-delNA virus showed a moderate growth advantage on avian cultured cells. In 4-week-old chickens inoculated intratracheally with the MZ-delNA virus, viral replication in the lungs, liver, and kidneys was enhanced and interstitial pneumonia lesions were more severe than with the wild-type virus. The MZ-delNA-inoculated chickens showed significantly increased levels of mRNAs encoding interleukin-6 (IL-6), transforming growth factor-β4 (TGF-β4), and CCL5 in the lungs and a higher frequency of apoptotic cells in the liver than did their MZ-inoculated counterparts. Molecular mechanisms possibly underlying the growth advantage of the MZ-delNA virus were explored. The measured enzymatic activities toward a small substrate were similar for the wild-type and deleted NA, but the MZ-delNA virus eluted from chicken erythrocytes at reduced rates. Pseudoviral particles expressing the MZ hemagglutinin in combination with the MZ-NA or MZ-delNA protein were produced from avian cultured cells with similar efficiencies, suggesting that the deletion in the NA stalk does not enhance the release of progeny virions and probably affects an earlier step of the viral cycle. Overall, our data indicate that a shortened NA stalk is a strong determinant of adaptation and virulence of waterfowl influenza viruses in chickens.

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Novel Ranking System for Identifying Efficacious Anti-Influenza Virus PB2 Inhibitors

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00781-15 ◽

2015 ◽

Vol 59 (10) ◽

pp. 6007-6016 ◽

Cited By ~ 8

Author(s):

Alice W. Tsai ◽

Colleen F. McNeil ◽

Joshua R. Leeman ◽

Hamilton B. Bennett ◽

Kwame Nti-Addae ◽

...

Keyword(s):

Influenza Virus ◽

Influenza A Virus ◽

Influenza A ◽

Therapeutic Agents ◽

Influenza Viruses ◽

Antigenic Drift ◽

Whole Body ◽

Infection Model ◽

Virus Infections

ABSTRACTThrough antigenic drift and shifts, influenza virus infections continue to be an annual cause of morbidity in healthy populations and of death among elderly and at-risk patients. The emergence of highly pathogenic avian influenza viruses such as H5N1 and H7N9 and the rapid spread of the swine-origin H1N1 influenza virus in 2009 demonstrate the continued need for effective therapeutic agents for influenza. While several neuraminidase inhibitors have been developed for the treatment of influenza virus infections, these have shown a limited window for treatment initiation, and resistant variants have been noted in the population. In addition, an older class of antiviral drugs for influenza, the adamantanes, are no longer recommended for treatment due to widespread resistance. There remains a need for new influenza therapeutic agents with improved efficacy as well as an expanded window for the initiation of treatment. Azaindole compounds targeting the influenza A virus PB2 protein and demonstrating excellentin vitroandin vivoproperties have been identified. To evaluate thein vivoefficacy of these PB2 inhibitors, we utilized a mouse influenza A virus infection model. In addition to traditional endpoints, i.e., death, morbidity, and body weight loss, we measured lung function using whole-body plethysmography, and we used these data to develop a composite efficacy score that takes compound exposure into account. This model allowed the rapid identification and ranking of molecules relative to each other and to oseltamivir. The ability to identify compounds with enhanced preclinical properties provides an opportunity to develop more-effective treatments for influenza in patients.

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A single dose vaccination with an elastase-dependent H1N1 live attenuated swine influenza virus protects pigs from challenge with 2009 pandemic H1N1 virus

Acta veterinaria ◽

10.2478/acve-2014-0002 ◽

2014 ◽

Vol 64 (1) ◽

pp. 10-23

Author(s):

Aleksandar Mašić ◽

Niziti Woldeab ◽

Carissa Embury-Hyatt ◽

Yan Zhou ◽

Shawn Babiuk

Keyword(s):

Influenza Virus ◽

Single Dose ◽

Influenza A Virus ◽

Neutralizing Antibodies ◽

Influenza A ◽

Swine Influenza Virus ◽

Swine Influenza ◽

Influenza Viruses ◽

Virus Infections ◽

Intranasal Route

Abstract The 2009 outbreak of H1N1 influenza A viruses in humans underscored the importance of pigs in influenza A virus evolution and the emergence of novel viruses with pandemic potential. In addition, influenza A virus infections continued to cause production losses in the agricultural industry resulting in a significant drop of profit. The primary method to control influenza A virus infections in pigs is through vaccination. Previously we demonstrated that two doses of an elastase-dependent live attenuated swine influenza virus administered by either the intratracheal or intranasal route can provide a high degree of protection in pigs against challenge with both homologous and different heterologous swine influenza viruses. Here we report the protection efficacy of a single dose elastase-dependent live attenuated swine influenza virus administered by the intranasal route against challenge with homologous subtypic H1N1 2009 pandemic swine-like influenza virus. Protection was observed in the absence of neutralizing antibodies specific for H1N1 2009 in sera.

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Temperature-Sensitive Live-Attenuated Canine Influenza Virus H3N8 Vaccine

Journal of Virology ◽

10.1128/jvi.02211-16 ◽

2016 ◽

Vol 91 (4) ◽

Cited By ~ 8

Author(s):

Aitor Nogales ◽

Laura Rodriguez ◽

Caroline Chauché ◽

Kai Huang ◽

Emma C. Reilly ◽

...

Keyword(s):

Influenza Virus ◽

Influenza A ◽

Influenza Viruses ◽

Influenza Vaccines ◽

Respiratory Pathogen ◽

Temperature Sensitive ◽

Wild Type ◽

Canine Influenza Virus ◽

Canine Influenza ◽

Better Than

ABSTRACT Canine influenza is a respiratory disease of dogs caused by canine influenza virus (CIV). CIV subtypes responsible for influenza in dogs include H3N8, which originated from the transfer of H3N8 equine influenza virus to dogs; and the H3N2 CIV, which is an avian-origin virus that adapted to infect dogs. Influenza infections are most effectively prevented through vaccination to reduce transmission and future infection. Currently, only inactivated influenza vaccines (IIVs) are available for the prevention of CIV in dogs. However, the efficacy of IIVs is suboptimal, and novel approaches are necessary for the prevention of disease caused by this canine respiratory pathogen. Using reverse genetics techniques, we have developed a live-attenuated CIV vaccine (LACIV) for the prevention of H3N8 CIV. The H3N8 LACIV replicates efficiently in canine cells at 33°C but is impaired at temperatures of 37 to 39°C and was attenuated compared to wild-type H3N8 CIV in vivo and ex vivo. The LACIV was able to induce protection against H3N8 CIV challenge with a single intranasal inoculation in mice. Immunogenicity and protection efficacy were better than that observed with a commercial CIV H3N8 IIV but provided limited cross-reactive immunity and heterologous protection against H3N2 CIV. These results demonstrate the feasibility of implementing a LAIV approach for the prevention and control of H3N8 CIV in dogs and suggest the need for a new LAIV for the control of H3N2 CIV. IMPORTANCE Two influenza A virus subtypes has been reported in dogs in the last 16 years: the canine influenza viruses (CIV) H3N8 and H3N2 of equine and avian origins, respectively. To date, only inactivated influenza vaccines (IIVs) are available to prevent CIV infections. Here, we report the generation of a recombinant, temperature-sensitive H3N8 CIV as a live-attenuated influenza vaccine (LAIV), which was attenuated in mice and dog tracheal, explants compared to CIV H3N8 wild type. A single dose of H3N8 LACIV showed immunogenicity and protection against a homologous challenge that was better than that conferred with an H3N8 IIV, demonstrating the feasibility of implementing a LAIV approach for the improved control of H3N8 CIV infections in dogs.

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