Improvement of the inactivated SARS-CoV-2 vaccine potency through formulation in alum/naloxone adjuvant; Robust T cell and anti-RBD IgG responses

Document Type : Original Article

Authors

1 Advanced Therapy Medicinal Product (ATMP) Department, Breast Cancer Research Center, Motamed Cancer Institute, Academic Center for Education, Culture and Research (ACECR), Tehran, Iran

2 Recombinant Vaccine Research Center, Tehran University of Medical Sciences, Tehran, Iran

3 Department of FMD Vaccine Production, Razi Vaccine & Serum Research Institute, Agricultural Research, Education & Extension Organization (AREEO), Karaj, Iran

4 Immunotherapy Group, The Institute of Pharmaceutical Science (TIPS), Tehran University of Medical Science, Tehran, Iran

Abstract

Objective(s): SARS-CoV-2, emerging as a major threat to public health, has to be controlled through vaccination. Naloxone (NLX), an opioid receptor antagonist, demonstrated its adjuvant activity for microbial vaccines. In this study, inactivated SARS-CoV-2 was developed in the Alum/NLX adjuvant to increase the potency of the inactivated SARS-CoV-2 vaccine. 
Materials and Methods: BALB/c mice were immunized on days 0 and 14 with inactivated SARS-CoV-2-Alum, -Alum + NLX 3 mg/kg, -Alum + NLX 10 mg/kg, and -Freund adjuvant, as well as PBS. IFN-γ and IL-4 cytokines and Granzyme-B release were assessed with ELISA. In addition, specific total IgG, IgG1/IgG2a isotypes, and ratio as well as anti-RBD IgG responses were assessed with an optimized ELISA. 
Results: SARS-CoV-2-Alum-NLX10 group showed a significant increase in the IFN-γ cytokine response versus SARS-CoV-2-Alum, SARS-CoV-2-Alum-NLX3, and PBS groups. The SARS-CoV-2-Alum-NLX3 group exhibited a significant decrease in IL-4 cytokine versus SARS-CoV-2-Alum. The mice immunized with SARS-CoV-2-Alum-NLX10 showed a significant increase in CTL activity versus SARS-CoV-2-Alum and PBS. In addition, mice immunized with SARS-CoV-2-Alum-NLX3, SARS-CoV-2-Alum-NLX10 and SARS-CoV-2-Freund demonstrated an increase in IgG response, as compared with SARS-CoV-2-Alum and PBS group. Furthermore, all formulations of SARS-CoV-2 vaccines could induce both IgG1 and IgG2a isotypes. But, the IgG2a/IgG1 ratio in SARS-CoV-2-Freund and SARS-CoV-2-Alum-NLX10 revealed an increase as compared with that of the SARS-CoV-2-Alum group. Anti-RBD IgG response in the SARS-CoV-2-Alum-NLX10 group showed a significant increase as compared with the Alum-based vaccine. 
Conclusion: Formulation of inactivated SARS-CoV-2 virus in NLX/alum adjuvant improved the potency of humoral and, especially, cellular responses.

Keywords

References

1. Shi Y, Wang G, Cai XP, Deng JW, Zheng L, Zhu HH, et al. An overview of COVID-19. J Zhejiang Univ Sci B 2020;21:343-360.
2. Chang L, Yan Y, Wang L. Coronavirus disease 2019: Coronaviruses and blood safety. Transfus Med Rev 2020;34:75-80.
3. The Lancet Infectious D. Challenges of coronavirus disease 2019. Lancet Infect Dis 2020;20:261.
4. Dhama K, Khan S, Tiwari R, Sircar S, Bhat S, Malik YS, et al. Coronavirus disease 2019-COVID-19. Clin Microbiol Rev 2020;33:1-48.
5. Alkandari D, Herbert JA, Alkhalaf MA, Yates C, Panagiotou S. SARS-CoV-2 vaccines: Fast track versus efficacy.  Lancet Microbe 2021;2:e89-e90.
6. Iqbal Yatoo M, Hamid Z, Parray OR, Wani AH, Ul Haq A, Saxena A, et al. COVID-19-recent advancements in identifying novel vaccine candidates and current status of upcoming SARS-CoV-2 vaccines. Hum Vaccin Immunother 2020;16:2891-2904. 
7. Dhama K, Sharun K, Tiwari R, Dadar M, Malik YS, Singh KP, et al. COVID-19, an emerging coronavirus infection: Advances and prospects in designing and developing vaccines, immunotherapeutics, and therapeutics. Human vaccines & immunotherapeutics. 2020;16:1232-1238.
8. Jafari A, Danesh Pouya F, Niknam Z, Abdollahpour-Alitappeh M, Rezaei-Tavirani M, Rasmi Y. Current advances and challenges in COVID-19 vaccine development: from conventional vaccines to next-generation vaccine platforms. Mol Biol Rep 2022;Vol:1-15. 
9. Najminejad H, Kalantar SM, Mokarram AR, Dabaghian M, Abdollahpour-Alitappeh M, Ebrahimi SM, et al. Bordetella pertussis antigens encapsulated into N-trimethyl chitosan nanoparticulate systems as a novel intranasal pertussis vaccine. Artif Cells Nanomed Biotechnol 2019;47:2605-2611.
10.    Amini Y, Tebianian M, Mosavari N, Fasihi Ramandi M, Ebrahimi SM, Najminejad H, et al. Development of an effective delivery system for intranasal immunization against Mycobacterium tuberculosis ESAT-6 antigen. Artif Cells Nanomed Biotechnol 2017;45:291-296.
11.    Corey L, Mascola JR, Fauci AS, Collins FS. A strategic approach to COVID-19 vaccine R&D. Science 2020;368:948-950.
12.    Mahdavi M, Ebtekar M, Khorshid HRK, Azadmanesh K, Hartoonian C, Hassan ZM. ELISPOT analysis of a new CTL based DNA vaccine for HIV-1 using GM-CSF in DNA prime/peptide boost strategy: GM-CSF induced long-lived memory responses. Immunology letters 2011;140:14-20.
13.    Wang J, Peng Y, Xu H, Cui Z, Williams RO. The COVID-19 vaccine race: challenges and opportunities in vaccine formulation. AAPS PharmSciTech 2020;21:1-12.
14.    Kuo T-Y, Lin M-Y, Coffman RL, Campbell JD, Traquina P, Lin Y-J, et al. Development of CpG-adjuvanted stable prefusion SARS-CoV-2 spike antigen as a subunit vaccine against COVID-19. Scientific Reports 2020;10:1-10.
15.    García-Arriaza J, Garaigorta U, Pérez P, Lázaro-Frías A, Zamora C, Gastaminza P, et al. COVID-19 vaccine candidates based on modified vaccinia virus ankara expressing the SARS-CoV-2 spike protein induce robust t-and b-cell immune responses and full efficacy in mice. J Virol 2021;95:e02260-20. 
16.    Cox RJ, Brokstad KA. Not just antibodies: B cells and T cells mediate immunity to COVID-19. Nat Rev Immunol 2020;20:581-582.
17.    Beyer WEP, Palache AM, Reperant LA, Boulfich M, Osterhaus A. Association between vaccine adjuvant effect and pre-seasonal immunity. Systematic review and meta-analysis of randomised immunogenicity trials comparing squalene-adjuvanted and aqueous inactivated influenza vaccines. Vaccine. 2020;38:1614-1622.
18.    Jamali A, Mahdavi M, Hassan ZM, Sabahi F, Farsani MJ, Bamdad T, et al. A novel adjuvant, the general opioid antagonist naloxone, elicits a robust cellular immune response for a DNA vaccine. Int Immunol 2009;21:217-225.
19.    Burris S, Norland J, Edlin BR. Legal aspects of providing naloxone to heroin users in the United States. International Journal of Drug Policy 2001;12:237-248.
20.    Yasaghi M, Mahdavi M. Potentiation of human papilloma vaccine candidate using naloxone/alum mixture as an adjuvant: increasing immunogenicity of HPV-16E7d vaccine. Iran J Basic Med Sci 2016; 19: 1003–1009. 
21.    Jamali A, Mahdavi M, Shahabi S, Hassan ZM, Sabahi F, Javan M, et al. Naloxone, an opioid receptor antagonist, enhances induction of protective immunity against HSV-1 infection in BALB/c mice. Microb Pathog 2007;43:217-223.
22.    Jazani NH, Karimzad M, Mazloomi E, Sohrabpour M, Hassan ZM, Ghasemnejad H, et al. Evaluation of the adjuvant activity of naloxone, an opioid receptor antagonist, in combination with heat-killed Listeria monocytogenes vaccine. Microbes Infect 2010;12:382-388.
23.    Velashjerdi Farahani S, Reza Aghasadeghi M, Memarnejadian A, Faezi S, Shahosseini Z, Mahdavi M, et al. Naloxone/alum mixture a potent adjuvant for HIV-1 vaccine: induction of cellular and poly-isotypic humoral immune responses. Pathog Glob Health 2016;110:39-47.
24.    Kaffashi A, Huang J, Bairami A, Fallah Mehrabadi MH, Yaslianifard S, Bashashati M, et al. Complete genome sequencing and molecular characterization of SARS-COV-2 from COVID-19 cases in Alborz province in Iran. Heliyon 2021;7:e08027.
25.    Fathi M, Nezamzadeh R, Abdollahpour‐Alitappeh M, Yazdi MH, Khoramabadi N, Mahdavi M. Formulation of a recombinant HIV‐1 polytope candidate vaccine with naloxone/alum mixture: Induction of multi‐cytokine responses with a higher regulatory mechanism. APMIS 2021;129:480-488. 
26.    Mojarab S, Shahbazzadeh D, Moghbeli M, Eshraghi Y, Bagheri KP, Rahimi R, et al. Immune responses to HIV-1 polytope vaccine candidate formulated in aqueous and alcoholic extracts of Propolis: Comparable immune responses to Alum and Freund adjuvants. Microbial Pathogenesis 2020;140:103932.
27.    Mahdavi M, Tajik AH, Ebtekar M, Rahimi R, Adibzadeh MM, Moozarmpour HR, et al. Granulocyte‐macrophage colony‐stimulating factor, a potent adjuvant for polarization to Th‐17 pattern: an experience on HIV‐1 vaccine model. Apmis. 2017;125:596-603.
28.    Pi-Estopiñan F, Pérez MT, Fraga A, Bergado G, Díaz GD, Orosa I, et al. A cell-based ELISA as surrogate of virus neutralization assay for RBD SARS-CoV-2 specific antibodies. Vaccine 2022;40:1958-1967.
29.    Hellewell J, Abbott S, Gimma A, Bosse NI, Jarvis CI, Russell TW, et al. Feasibility of controlling COVID-19 outbreaks by isolation of cases and contacts. Lancet Global Health 2020;8:e488-e96.
30.    Forni G, Mantovani A. Covid-19 vaccines: Where we stand and challenges ahead. Cell Death Differ 2021;28:626-639.
31.    Liu X, Liu C, Liu G, Luo W, Xia N. Covid-19: Progress in diagnostics, therapy and vaccination. Theranostics 2020;10:7821-7835.
32.    Lazarus JV, Ratzan SC, Palayew A, Gostin LO, Larson HJ, Rabin K, et al. A global survey of potential acceptance of a Covid-19 vaccine. Nat Med 2021;27:225-228.
33.    Kim JH, Marks F, Clemens JD. Looking beyond Covid-19 vaccine phase 3 trials. Nat Med 2021;27:205-211.
34.    Jazani NH, Parsania S, Sohrabpour M, Mazloomi E, Karimzad M, Shahabi S. Naloxone and alum synergistically augment adjuvant activities of each other in a mouse vaccine model of Salmonella typhimurium infection. Immunobiology 2011;216:744-751.
35.    Khorshidvand Z, Shahabi S, Mohamadzade H, Daryani A, Tappeh KH. Mixture of alum–naloxone and alum–naltrexone as a novel adjuvant elicits immune responses for Toxoplasma gondii lysate antigen in BALB/c mice. Experimental Parasitology 2016;162:28-34.
36.    Sacerdote P, Gaspani L, Panerai AE. The opioid antagonist naloxone induces a shift from type 2 to type 1 cytokine pattern in normal and skin‐grafted mice. Ann N Y Acad Sci 2000;917:755-763.
37.    Hassan ATM, Hassan ZM, Moazzeni SM, Mostafaie A, Shahabi S, Ebtekar M, et al. Naloxone can improve the anti-tumor immunity by reducing the CD4+ CD25+ Foxp3+ regulatory T cells in BALB/c mice. Int Immunopharmacol 2009;9:1381-1386.
38.    Rostami H, Ebtekar M, Ardestani MS, Yazdi MH, Mahdavi M. Co-utilization of a TLR5 agonist and nano-formulation of HIV-1 vaccine candidate leads to increased vaccine immunogenicity and decreased immunogenic dose: A preliminary study. Immunol Lett 2017;187:19-26.
39.    Jazani NH, Sohrabpour M, Mazloomi E, Shahabi SJFI, Microbiology M. A novel adjuvant, a mixture of alum and the general opioid antagonist naloxone, elicits both humoral and cellular immune responses for heat-killed Salmonella typhimurium vaccine. FEMS Immunol Med Microbiol 2011;61:54-62.
40.    Caielli S, Veiga DT, Balasubramanian P, Athale S, Domic B, Murat E, et al. A CD4+ T cell population expanded in lupus blood provides B cell help through interleukin-10 and succinate. Nat Med 2019;25:75-81.
41.    Kim ST, Choi J-Y, Lainez B, Schulz VP, Karas DE, Baum ED, et al. Human extrafollicular CD4+ Th cells help memory B cells produce Igs. J Immunol 2018;201:1359-1372.
42.    Jazani NH, Parsania S, Sohrabpour M, Mazloomi E, Karimzad M, Shahabi SJI. Naloxone and alum synergistically augment adjuvant activities of each other in a mouse vaccine model of Salmonella typhimurium infection. Immunobiology 2011;216:744-751.
43.    Khoury DS, Cromer D, Reynaldi A, Schlub TE, Wheatley AK, Juno JA, et al. Neutralizing antibody levels are highly predictive of immune protection from symptomatic SARS-CoV-2 infection. Nat Med 2021;27:1205-1211. 
44.    Starr TN, Czudnochowski N, Liu Z, Zatta F, Park YJ, Addetia A, et al. SARS-CoV-2 RBD antibodies that maximize breadth and resistance to escape. Nature 2021 Sep;597:97-102.
45.    Sauer K, Harris T. An Effective COVID-19 Vaccine Needs to Engage T Cells. Front Immunol 2020;11:581807. 
46.    Cañete PF, Vinuesa CG. COVID-19 makes B cells forget, but T cells remember. Cell 2020;183:13-15.
Iranian Journal of Basic Medical Sciences
Volume 25, Issue 5
May 2022
Pages 554-561

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