Sulfur-containing class of broad-spectrum antivirals improves influenza virus vaccine development

1. Prioritizing diseases for research and development in emergency contexts. Available from: https://www.who.int/activities/prioritizing-diseases-for-research-and-development-in-emergency-contexts (2021).

2. Paramore, L. C. et al. Economic impact of respiratory syncytial virus-related illness in the US: an analysis of national databases. Pharmacoeconomics 22, 275–284 (2004).

   Google Scholar 

3. Fendrick, A. M. et al. The economic burden of non-influenza-related viral respiratory tract infection in the United States. Arch. Intern. Med. 163, 487–494 (2003).

   Google Scholar 

4. Frank, T. D. Global, regional, and national incidence, prevalence, and mortality of HIV, 1980-2017, and forecasts to 2030, for 195 countries and territories: a systematic analysis for the Global Burden of Diseases, Injuries, and Risk Factors Study 2017. Lancet HIV 6, e831–e859 (2019).

   Google Scholar 

5. Excler, J. L. et al. Vaccine development for emerging infectious diseases. Nat. Med. 27, 591–600 (2021).

   Google Scholar 

6. Sridhar, S., Brokstad, K. A. & Cox, R. J. Influenza vaccination strategies: comparing inactivated and live attenuated influenza vaccines. Vaccines 3, 373–389 (2015).

   Google Scholar 

7. Vartak, A. & Sucheck, S. J. Recent advances in subunit vaccine carriers. Vaccines 4, 12 (2016).

8. Delrue, I. et al. Inactivated virus vaccines from chemistry to prophylaxis: merits, risks and challenges. Expert Rev. Vaccines 11, 695–719 (2012).

   Google Scholar 

9. Killikelly, A. M., Kanekiyo, M. & Graham, B. S. Pre-fusion F is absent on the surface of formalin-inactivated respiratory syncytial virus. Sci. Rep. 6, 34108 (2016).

   Google Scholar 

10. Fan, Y. C. et al. Formalin inactivation of Japanese encephalitis virus vaccine alters the antigenicity and immunogenicity of a neutralization epitope in envelope protein domain III. PLoS Negl. Trop. Dis. 9, e0004167 (2015).

    Google Scholar 

11. Chowdhury, P. et al. Comparison of β-propiolactone and formalin inactivation on antigenicity and immune response of West Nile virus. Adv. Virol. 2015, 616898 (2015).

    Google Scholar 

12. Jonges, M. et al. Influenza virus inactivation for studies of antigenicity and phenotypic neuraminidase inhibitor resistance profiling. J. Clin. Microbiol. 48, 928–940 (2010).

    Google Scholar 

13. Moghaddam, A. et al. A potential molecular mechanism for hypersensitivity caused by formalin-inactivated vaccines. Nat. Med. 12, 905–907 (2006).

    Google Scholar 

14. Murphy, B. R. & Walsh, E. E. Formalin-inactivated respiratory syncytial virus vaccine induces antibodies to the fusion glycoprotein that are deficient in fusion-inhibiting activity. J. Clin. Microbiol. 26, 1595–1597 (1988).

    Google Scholar 

15. Herrera-Rodriguez, J. et al. Inactivated or damaged? Comparing the effect of inactivation methods on influenza virions to optimize vaccine production. Vaccine 37, 1630–1637 (2019).

    Google Scholar 

16. Astill, J. et al. Examination of the effects of virus inactivation methods on the induction of antibody- and cell-mediated immune responses against whole inactivated H9N2 avian influenza virus vaccines in chickens. Vaccine 36, 3908–3916 (2018).

    Google Scholar 

17. Vaccine Effectiveness: How Well Do the Flu Vaccines Work?; Available from: https://www.cdc.gov/flu/vaccines-work/vaccineeffect.htm?web=1&wdLOR=c8D59CC9A-979E-3A4D-82E3-7AEF3D351FDF (2020).

18. Up to 650 000 people die of respiratory diseases linked to seasonal flu each year; Available from: https://www.who.int/mediacentre/news/statements/2017/flu/en/ (2017).

19. Kon, T. C. et al. Influenza vaccine manufacturing: effect of inactivation, splitting and site of manufacturing. Comparison of influenza vaccine production processes. PLoS ONE 11, e0150700 (2016).

    Google Scholar 

20. Mazur, N. I. et al. The respiratory syncytial virus vaccine landscape: lessons from the graveyard and promising candidates. Lancet Infect. Dis. 18, e295–e311 (2018).

    Google Scholar 

21. Kapikian, A. Z. et al. An epidemiologic study of altered clinical reactivity to respiratory syncytial (RS) virus infection in children previously vaccinated with an inactivated RS virus vaccine. Am. J. Epidemiol. 89, 405–421 (1969).

    Google Scholar 

22. Leyssen, P. et al. The predominant mechanism by which ribavirin exerts its antiviral activity in vitro against flaviviruses and paramyxoviruses is mediated by inhibition of IMP dehydrogenase. J. Virol. 79, 1943–1947 (2005).

    Google Scholar 

23. Vigant, F. et al. A mechanistic paradigm for broad-spectrum antivirals that target virus-cell fusion. PLoS Pathog. 9, e1003297 (2013).

24. Li, H. et al. Role of hydrogen sulfide in paramyxovirus infections. J. Virol. 89, 5557–5568 (2015).

    Google Scholar 

25. Park, C. M. et al. Synthesis and evaluation of phosphorodithioate-based hydrogen sulfide donors. Mol. Biosyst. 9, 2430–2434 (2013).

    Google Scholar 

26. Iciek, M., Kwiecień, I. & Włodek, L. Biological properties of garlic and garlic-derived organosulfur compounds. Environ. Mol. Mutagen. 50, 247–265 (2009).

    Google Scholar 

27. Wolf, M. C. et al. A broad-spectrum antiviral targeting entry of enveloped viruses. Proc. Natl. Acad. Sci. USA 107, 3157–3162 (2010). p.

    Google Scholar 

28. Aguilar, H. C. et al. N-glycans on Nipah virus fusion protein protect against neutralization but reduce membrane fusion and viral entry. J. Virol. 80, 4878–4889 (2006).

    Google Scholar 

29. Aguilar, H. C. et al. Polybasic KKR motif in the cytoplasmic tail of Nipah virus fusion protein modulates membrane fusion by inside-out signaling. J. Virol. 81, 4520–4532 (2007).

    Google Scholar 

30. Wolf, M. C. et al. A catalytically and genetically optimized beta-lactamase-matrix based assay for sensitive, specific, and higher throughput analysis of native henipavirus entry characteristics. Virol. J. 6, 119 (2009).

    Google Scholar 

31. Landowski, M. et al. Nipah virion entry kinetics, composition, and conformational changes determined by enzymatic virus-like particles and new flow virometry tools. J. Virol. 88, 14197–14206 (2014).

    Google Scholar 

32. Aguilar, H. C. et al. A novel receptor-induced activation site in the Nipah virus attachment glycoprotein (G) involved in triggering the fusion glycoprotein (F). J. Biol. Chem. 284, 1628–1635 (2009).

    Google Scholar 

33. Liu, Q. et al. Nipah virus attachment glycoprotein stalk C-terminal region links receptor binding to fusion triggering. J. Virol. 89, 1838–1850 (2015).

    Google Scholar 

34. Lai, A. L. et al. The SARS-CoV fusion peptide forms an extended bipartite fusion platform that perturbs membrane order in a calcium-dependent manner. J. Mol. Biol. 429, 3875–3892 (2017).

    Google Scholar 

35. Lai, A. L. & Freed, J. H. The interaction between influenza HA fusion peptide and transmembrane domain affects membrane structure. Biophys. J. 109, 2523–2536 (2015).

    Google Scholar 

36. Lai, A. L. & Freed, J. H. HIV gp41 fusion peptide increases membrane ordering in a cholesterol-dependent fashion. Biophys. J. 106, 172–181 (2014).

    Google Scholar 

37. Ge, M. T. & Freed, J. H. Two conserved residues are important for inducing highly ordered membrane domains by the transmembrane domain of influenza hemagglutinin. Biophys. J. 100, 90–97 (2011).

    Google Scholar 

38. Ge, M. & Freed, J. H. Fusion peptide from influenza hemagglutinin increases membrane surface order: an electron-spin resonance study. Biophys. J. 96, 4925–4934 (2009).

    Google Scholar 

39. Liang, Z. C. & Freed, J. H. An assessment of the applicability of multifrequency ESR to study the complex dynamics of biomolecules. J. Phys. Chem. B 103, 6384–6396 (1999).

    Google Scholar 

40. Danieli, T. et al. Membrane fusion mediated by the influenza virus hemagglutinin requires the concerted action of at least three hemagglutinin trimers. J. Cell Biol. 133, 559–569 (1996).

    Google Scholar 

41. Budil, D. E. et al. Nonlinear-least-squares analysis of slow-motion EPR spectra in one and two dimensions using a modified Levenberg-Marquardt algorithm. J. Magn. Reson. Ser. A 120, 155–189 (1996).

    Google Scholar 

42. Bianco, C. L. et al. The chemical biology of the persulfide (RSSH)/perthiyl (RSS·) redox couple and possible role in biological redox signaling. Free Radic. Biol. Med. 101, 20–31 (2016).

    Google Scholar 

43. Park, C. M. et al. Persulfides: current knowledge and challenges in chemistry and chemical biology. Mol. Biosyst. 11, 1775–1785 (2015).

    Google Scholar 

44. Meher, G. & Chakraborty, H. Membrane composition modulates fusion by altering membrane properties and fusion peptide structure. J. Membr. Biol. 252, 261–272 (2019).

    Google Scholar 

45. Gibellini, F. & Smith, T. K. The Kennedy pathway-de novo synthesis of phosphatidylethanolamine and phosphatidylcholine. IUBMB Life 62, 414–428 (2010).

    Google Scholar 

46. Harris, K. et al. Intramuscular immunization of mice with live influenza virus is more immunogenic and offers greater protection than immunization with inactivated virus. Virol. J. 8, 251 (2011).

    Google Scholar 

47. Szretter, K. J., Balish, A. L. & Katz, J. M. Influenza: propagation, quantification, and storage. Curr. Protoc. Microbiol. Chapter 15, Unit 15G.1 (2006).

48. Leang, S. K. & Hurt, A. C. Fluorescence-based neuraminidase inhibition assay to assess the susceptibility of influenza viruses to the neuraminidase inhibitor class of antivirals. Jove-J. Vis. Exp. 122, 7 (2017).

    Google Scholar 

49. Wohlbold, T. J. et al. Vaccination with adjuvanted recombinant neuraminidase induces broad heterologous, but not heterosubtypic, cross-protection against influenza virus infection in mice. mBio 6, e02556 (2015).

    Google Scholar 

50. Trammell, R. A. & Toth, L. A. Markers for predicting death as an outcome for mice used in infectious disease research. Comp. Med. 61, 492–498 (2011).

    Google Scholar 

51. Hankaniemi, M. M. et al. A comparative study of the effect of UV and formalin inactivation on the stability and immunogenicity of a Coxsackievirus B1 vaccine. Vaccine 37, 5962–5971 (2019).

    Google Scholar 

52. Chowdhury, P. et al. Comparison of beta-propiolactone and formalin inactivation on antigenicity and immune response of West Nile virus. Adv. Virol. 2015, 616898 (2015).

53. Wilton, T. et al. Effect of formaldehyde inactivation on poliovirus. J. Virol. 88, 11955–11964 (2014).

    Google Scholar 

54. Ohki, S. & Arnold, K. Experimental evidence to support a theory of lipid membrane fusion. Colloids Surf. B Biointerfaces 63, 276–281 (2008).

    Google Scholar 

55. Cooper, S. T. & McNeil, P. L. Membrane repair: mechanisms and pathophysiology. Physiol. Rev. 95, 1205–1240 (2015).

    Google Scholar 

56. Krammer, F. The human antibody response to influenza A virus infection and vaccination. Nat. Rev. Immunol. 19, 383–397 (2019). p.

    Google Scholar 

57. Möller, L. et al. Evaluation of virus inactivation by formaldehyde to enhance biosafety of diagnostic electron microscopy. Viruses 7, 666–679 (2015).

    Google Scholar 

58. Fink, A. L. et al. Biological sex affects vaccine efficacy and protection against influenza in mice. Proc. Natl. Acad. Sci. USA 115, 12477–12482 (2018).

    Google Scholar 

59. Tan, G. S. et al. Broadly-reactive neutralizing and non-neutralizing antibodies directed against the H7 influenza virus hemagglutinin reveal divergent mechanisms of protection. PLoS Pathog. 12, e1005578 (2016).

    Google Scholar 

60. Zhao, Y., Biggs, T. D. & Xian, M. Hydrogen sulfide (H2S) releasing agents: chemistry and biological applications. Chem. Commun. 50, 11788–11805 (2014).

    Google Scholar 

61. Pinello, J. F. et al. Structure-function studies link class ii viral fusogens with the ancestral gamete fusion protein HAP2. Curr. Biol. 27, 651–660 (2017).

    Google Scholar 

62. Tran, A. et al. Rapid detection of viral envelope lipids using lithium adducts and AP-MALDI high-resolution mass spectrometry. J. Am. Soc. Mass Spectrom 32, 2322–2333 (2021).

63. Tran, A. et al. Lithium hydroxide hydrolysis combined with MALDI TOF mass spectrometry for rapid sphingolipid detection. J. Am. Soc. Mass Spectrom. 32, 289–300 (2021).

    Google Scholar 

64. Tang, T. et al. Functional infectious nanoparticle detector: finding viruses by detecting their host entry functions using organic bioelectronic devices. ACS Nano 15, 18142–18152 (2021).

    Google Scholar 

65. Hagen, W. J. H., Wan, W. & Briggs, J. A. G. Implementation of a cryo-electron tomography tilt-scheme optimized for high resolution subtomogram averaging. J. Struct. Biol. 197, 191–198 (2017).

    Google Scholar 

66. Eisenstein, F. et al. Parallel cryo electron tomography on in situ lamellae. Nat. Methods 20, 131–138 (2023).

    Google Scholar 

67. Mastronarde, D. N. Automated electron microscope tomography using robust prediction of specimen movements. J. Struct. Biol. 152, 36–51 (2005).

    Google Scholar 

68. Zheng, S. Q. et al. MotionCor2: anisotropic correction of beam-induced motion for improved cryo-electron microscopy. Nat. Methods 14, 331–332 (2017).

    Google Scholar 

69. Kremer, J. R., Mastronarde, D. N. & McIntosh, J. R. Computer visualization of three-dimensional image data using IMOD. J. Struct. Biol. 116, 71–76 (1996).

    Google Scholar 

70. Hardy, M. E. et al. 18β-glycyrrhetinic acid inhibits rotavirus replication in culture. Virol. J. 9, 96 (2012).

    Google Scholar 

71. McKimm-Breschkin, J. L. A simplified plaque assay for respiratory syncytial virus-direct visualization of plaques without immunostaining. J. Virol. Methods 120, 113–117 (2004).

    Google Scholar 

72. Lieber, D. & Bailer, S. M. Determination of HSV-1 infectivity by plaque assay and a luciferase reporter cell line. Methods Mol. Biol. 1064, 171–181 (2013).

    Google Scholar 

73. Huang, W. S. et al. ITK signalling via the Ras/IRF4 pathway regulates the development and function of Tr1 cells. Nat. Commun. 8, 15 (2017).

    Google Scholar 

Download references

Author notes

1. These authors contributed equally: David W. Buchholz, Armando Pacheco.

Authors and Affiliations

1. Department of Microbiology and Immunology, College of Veterinary Medicine, Cornell University, Ithaca, NY, USA

   David W. Buchholz, Armando Pacheco, I. Abrrey Monreal, Brian Imbiakha, Julie Sahler, Mason Jager, Erik M. Contreras, Shahrzad Ezzatpour, Yao Yu Yeo, Andrew Ma, Haewon Byun, Obaed Shah, J. Lizbeth Reyes Zamora, Gerlinde R. Van de Walle, Avery August & Hector C. Aguilar

2. Department of Chemistry, College of Arts and Sciences, Washington State University, Pullman, WA, USA

   Armando Pacheco, Shi Xu, Brandan Cook, Chuntao Yang, Yu Zhao & Ming Xian

3. Robert Frederick Smith School of Chemical and Biomolecular Engineering, College of Engineering, Cornell University, Ithaca, NY, USA

   Sreetama Pal, Zeinab J. Mohamed, Cheyan Xu & Susan Daniel

4. Department of Chemistry, Brown University, Providence, RI, USA

   Shi Xu & Ming Xian

5. Department of Chemistry and Chemical Biology, College of Arts and Sciences, Cornell University, Ithaca, NY, USA

   Alex Liqi Lai & Jack H. Freed

6. Department of Chemistry and Biochemistry, University of California, Los Angeles, CA, USA

   Elshan Ralalage & Michael J. Jung

7. Paul G. Allen School for Global Animal Health, College of Veterinary Medicine, Washington State University, Pullman, WA, USA

   Qian Liu

8. Department of Veterinary Microbiology and Pathology, College of Veterinary Medicine, Washington State University, Pullman, WA, USA

   Niraj K. Shil, Suzanne M. Pritchard, Santanu Bose & Anthony V. Nicola

9. Department of Biology, Indiana University, Bloomington, IN, USA

   Sara Jones-Burrage & Suchetana Mukhopadhyay

10. Center for Vaccines and Immunity, The Research Institute at Nationwide Children’s Hospital, Columbus, OH, USA

    Masako Shimamura

11. School of Molecular Biosciences, College of Veterinary Medicine, Washington State University, Pullman, WA, USA

    Alan G. Goodman

12. College of Letters, Sciences and Professional Studies, Montana Technological University, Butte, MT, USA

    Michele Hardy

13. Department of Virology, Center for Infectious Diseases, Uni-Heidelberg University Hospital, Heidelberg, Germany

    Petr Chlanda

14. Department of Pharmaceutical Sciences, School of Pharmacy, University of Maryland, Baltimore, MD, USA

    Jace W. Jones

15. Department of Microbiology, Immunology, and Molecular Genetics, David Geffen School of Medicine and College of Life Sciences, University of California, Los Angeles, CA, USA

    Hector C. Aguilar

Contributions

D.W.B., A.P., S.P., I.A.M., S.X., B.I., J.S., M.J., A.L.L., E.M.C., S.E., B.C., E.R., Q.L., Y.Y.Y., A.M., H.B., O.S., J.L.Z., N.K.S., S.J.-B., S.M.P., C.Y., Y.Z., Z.J.M., C.X., M.J.J., G.V.d.W., S.M., M.S., A.G.G., M.H., S.B., A.V.N., J.H.F., A.A., S.D., P.C., J.W.J., M.X. and H.C.A. designed and/or performed experiments. D.W.B., A.P., S.P., I.A.M., E.M.C., J.W.J., P.C. and H.A.C. wrote the manuscript and D.W.B., A.P., S.P., I.A.M., J.L.Z., S.D., M.J.J., G.V.d.W., S.M., M.S., A.G.G., A.A., J.W.J., M.H., S.B., A.V.N., J.H.F., P.C., M.X. and H.A.C. edited the manuscript.

Corresponding author

Correspondence to Hector C. Aguilar.

Competing interests

HAC, DWB, and IAM are inventors on patent applications related to XM-01 antivirals and XM-01–generated vaccines filed by Cornell University. The remaining authors declare no competing interests.

Peer review information

Nature Communications thanks Amir Ghaemi and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. A peer review file is available.

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

Reprints and permissions