Outer membrane vesicles associated with Dimethyldioctadecylammonium bromide or Saponin: protocols for antigens and adjuvants

Outer membrane vesicles associated with Dimethyldioctadecylammonium bromide or Saponin: protocols for antigens and adjuvants

Amanda Izeli Portilho^1,2, Gabrielle Gimenes Lima^1,2, Elizabeth De Gaspari^1,2 

¹Immunology Center, Adolfo Lutz Institute, São Paulo, SP, Brazil

²Graduate Program Interunits in Biotechnology, University of São Paulo, São Paulo, SP, Brazil

Correspondence to: Elizabeth De Gaspari

Immunology Center, Adolfo Lutz Institute. Av Dr Arnaldo, 355, 11th floor, room 1116, Cerqueira César, São Paulo, SP, Brazil. Postal code: 01246-902. Telephone: 55 11 30682898. Email: elizabeth.gaspari@ial.sp.gov.br

1. Introduction

Our group has been studying Dimethyldioctadecylammonium bromide (DDA) and Saponin (Sap) as adjuvants for outer membrane vesicles (OMVs) from Neisseria meningitidis, Neisseria lactamica and, more recently, for SARS-CoV-2 recombinant antigen [1–8]. Here, we describe the protocols used to obtain the Outer membrane vesicles (OMVs) of Neisseria and the adjuvants.

2. Outer membrane vesicles

The outer membrane vesicles (OMVs) are vesicles naturally released by gram negative bacteria, through the blebbing of their complex outer membrane [9]. The OMVs carry several antigens, which can trigger an specific immune response to the pathogen or activate the innate immunity after recognition of pattern-recognition receptors (PRRs) [10]. There are licensed OMV vaccines against Neisseria meningitidis [11], but considering the immunity activation, there are studies successfully using them as adjuvants for robust humoral and cellular responses [12–14].

2.1 OMVs Protocol

1. Culture the Neisseria strain in Tryptic Soy Broth (TSB) agar supplemented with 10% heat-inactivated horse serum, at 37ºC and 5% CO₂, for 24 hours;
2. Cover the Petri dishes with a 0.1 M sodium acetate 0.2 M lithium chloride solution (pH 5.8), gently releasing the colonies using a Drigalski spatula;
3. Pippete the suspension and transfer it to a tube containing 2 mm glass beads and incubate it in a shaker at 45ºC, 300 rpm, for 2 hours
4. Collect the supernatant and centrifugate at 4ºC, 12,000 rpm, for 20 minutes;
5. Dialyze the supernatant in 0.15 M NaCl solution, at 4ºC, overnight;
6. Concentrate the OMVs resting the dialysis membranes in PEG 8000 for 1 to 2 hours;
7. Dialyze the OMVs again (step 4).

It is important to highlight that OMVs contain LPS. There is an acceptable level of LPS for preclinical use [15], so it might be necessary to detoxify the OMVs from LPS, using Polimyxin B-Sepharose columns, for example, or another suitable protocol. Moreover, different OMV extraction protocols result in different OMV content, which is likely to affect the immunogenic potential [16].

3. Dimethyldioctadecylammonium bromide (DDA and DDA-BF)

DDA is a vesicular-formed cationic lipid with adjuvant properties. Besides encapsulating the antigen, protecting it from degradation, it promotes depot effect and favors interaction with antigen presenting cells – while the cells are negative charged, the adjuvant is positive-charged [17,18]. Another option to use DDA is obtaining bilayer fragments (DDA-BF) after sonication of the vesicles. The procedure results in a better stability and provides a larger interaction surface for the antigen [19]. It is employed in the commercial adjuvant Cationic formulation (CAF) 01 [20].

Our group has been working with DDA for over a decade, mostly using Neisseria antigens. The adjuvant contributed for cross-reactive responses, provided that immunization with N. lactamica resulted in recognition of N. meningitidis antigens and high antibody titers since the first dose of immunization [1]. Thus, it supported the maternal-fetal transference of antibodies [2] and t was a promising adjuvant for middle-aged mice, increasing immunogenicity and supporting humoral and cellular responses [6]. When OMV+DDA-BF were administrated via subcutaneous and intramuscular routes, we could not verify significant differences [5]. However, the adjuvant is also promising for mucosal immunization [3].

Passive transference of antibodies to neonates, mucosal immunity and robust immunogenicity in the elderly are challenges that vaccinology must address to ensure adequate protection for all populations and effective disease-control [21–23]. Taken all that into consideration, our results suggest that DDA is a promising adjuvant option.

3.1 DDA Protocol

1. Prepare sterile saline 1 mM

2. Prepare DDA for a 2 mM concentration (stock solution)

3. Dilute the stock solution to prepare a 0.1 mM solution for use

4. Filter the solution using a 0.22 µm filter

5. Dilute the antigen in the DDA 0.1 mM and let them interact for 1 hour before the innoculation

3.2 DDA-BF Protocol

6. Prepare DDA as the steps 1—3 above

7. Take the DDA 0.1 mM to the sonicator at 25% amplitude for 15 minutes

8. Centrifuge the DDA-BF at 4ºC, 10,000 rpm, for 30 minutes (to separate the titanium from the sonicator probe)

9. Carefully transfer the DDA-BF to a new tube, avoiding to mix the titanium pellet

10. Filter the solution using a 0.22 µm filter

11. Dilute the antigen in the DDA-BF 0.1 mM and let them interact for 1 hour before the innoculation

In our laboratory, we verified that DDA/DDA-BF at 0.1 mM provides good adjuvancity; however, other concentrations can be tested using Zeta potential (item 4.1).

4. Saponin

Saponin is an immunostimulatory molecule extracted from the bark of Quillaja saponaria Molina tree, capable of eliciting robust cellular responses. Commercial adjuvants also use Sap, for example, the ISCOM (Immunostimulating complex) and the AS (Adjuvant system) 01 [24].

In our experience, OMVs+Sap resulted not only in robust cellular response, but also in a potent humoral response, mediated by all IgG isotypes, recognition of several Neisseria antigens and immunologic memory [7]. When mixed with DDA and a recombinant protein from SARS-CoV-2, it elicited neutralizing antibodies, high avidity IgG1, IgG2a and IgG2b, persistence of the humoral response and supported maternal-fetal transference of antibodies [8].

4.1 Saponin protocol

1. Prepare sterile saline 1 mM
2. Dilute the Saponin for a 1 mg/mL stock solution
3. In our laboratory, we verified that Saponin ranging from 1 to 10 µg/mL concentration is safe for use. The hemolysis test will be described below (item 4.2).

5. Additional information: structural characterization and toxicity

Two other protocols should be used to assess the better concentrations of DDA/DDA-BF and Saponin for use in each laboratory and experimental conditions. Zeta potential characterizes the size, charge and polydispersion index of the particles [3,7]. The desirable particle would be ranging from 20 to 400 nm, which is ideal for lymphatic drainage and phagocytosis by antigen presenting cells (APC) [25]. The polydispersion infers the stability of the particle, and indexes < 0.3 are considered stable [26]. Finally, APCs present slightly negative charges and cationic vesicles would improve the attraction and facilitate phagocytosis; however, there are studies showing that the charge did not affect the phagocytosis as much as size [27]. Performing the Zeta potential will support scientists to set the better adjuvant and/or antigen concentrations for size and stability of the vesicles.

Another test that should be proceeded is the hemolysis test [28]. Saponins are known to provoke hemolysis because of their amphiphilic structure, self- aggregation and capacity to interact with membrane compounds [29]. That considered, it is important to conduct a hemolysis test to assess the safest Saponin concentration for use. We use the protocol proposed by Pabreja et al. [28], which is simple to perform and analyze, suitable for most laboratories.

6. Conclusion

DDA and Saponin are being studied and the scientific evidence suggests them to be promising candidates for adjuvants, as well as Neisseria OMVs. We briefly described simple protocols to obtain these adjuvants and encourage studies using them. New adjuvants are should be studied, aiming two points: i) formulate vaccines that modulate the response according to the ideal pattern to control the pathogen – for example, humoral, cellular or mixed; and ii) support all countries manufacture of their own vaccines, which will improve the cost and consequentially, the access to vaccines.

Acknowledgements

The laboratory team over the last years: Adriana Freitas de Almeida, Caroline Argentati Gonçalves, Fabiana Mahylowski Rinaldi, Fernanda Ayane de Oliveira Santos, Gabriela Trzewikoswki de Lima, Luciana Tendolini Brito, Victor Araujo Correa and Emanuelle Baldo Gaspar.

Dr Nilton Lincopan, from the Microbiology Department of the Biomedical Sciences Institute, University of São Paulo, for support in DDA-BF preparation and Zeta potential analysis throughout these studies.

Dr Ana Paula Lemos, from the Bacteriology Center of Adolfo Lutz Institute and the Microorganisms Collection from Adolfo Lutz Institute for support in the isolation of Neisseria strains.

Financial support

These studies were supported by Fundação de Amparo à Pesquisa do Estado de São  Paulo  (FAPESP) (13/11147-2,  12/15568–0, 14/07182-0, 18/04202-0, 19/02042-9), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) (132743/2015-9; 132743/2014-6, 131412/2019–1, 131308/2021–1) and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) (88887.661236/2022–00).

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