Next Article in Journal
Predictors of Willingness of the General Public to Receive a Second COVID-19 Booster Dose or a New COVID-19 Vaccine: A Cross-Sectional Study in Greece
Next Article in Special Issue
Long-Term Longitudinal Analysis of Neutralizing Antibody Response to Three Vaccine Doses in a Real-Life Setting of Previously SARS-CoV-2 Infected Healthcare Workers: A Model for Predicting Response to Further Vaccine Doses
Previous Article in Journal
COVID-19 Vaccine Hesitancy: The Perils of Peddling Science by Social Media and the Lay Press
Previous Article in Special Issue
Vulnerability Predictors of Post-Vaccine SARS-CoV-2 Infection and Disease—Empirical Evidence from a Large Population-Based Italian Platform
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
Vaccines
Volume 10
Issue 7
10.3390/vaccines10071060
vaccines-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles thumb_up
...
Endorse textsms
...
Comment
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

Activation of Anti-SARS-CoV-2 Human CTLs by Extracellular Vesicles Engineered with the N Viral Protein

by
Francesco Manfredi
1,
Chiara Chiozzini
1,
Flavia Ferrantelli
1,
Patrizia Leone
1,
Andrea Giovannelli
2,
Massimo Sanchez
3 and
Maurizio Federico
1,*
1
National Center for Global Health, Istituto Superiore di Sanità, Viale Regina Elena 299, 00161 Rome, Italy
2
National Center for Animal Experimentation and Welfare, Istituto Superiore di Sanità, Viale Regina Elena 299, 00161 Rome, Italy
3
Core Facilities, Istituto Superiore di Sanità, Viale Regina Elena 299, 00161 Rome, Italy
*
Author to whom correspondence should be addressed.
Vaccines 2022, 10(7), 1060; https://doi.org/10.3390/vaccines10071060
Submission received: 24 May 2022 / Revised: 22 June 2022 / Accepted: 28 June 2022 / Published: 30 June 2022
(This article belongs to the Special Issue Vaccine Candidate against SARS-CoV-2)
Browse Figures
Review Reports Versions Notes

Abstract

:
We propose an innovative anti-SARS-CoV-2 immune strategy based on extracellular vesicles (EVs) inducing an anti-SARS-CoV-2 N CD8+ T cytotoxic lymphocyte (CTL) immune response. We previously reported that the SARS-CoV-2 N protein can be uploaded at high levels in EVs upon fusion with Nefmut, i.e., a biologically inactive HIV-1 Nef mutant incorporating into EVs at quite high levels. Here, we analyze the immunogenic properties in human cells of EVs engineered with SARS-CoV-2 N fused at the C-terminus of either Nefmut or a deletion mutant of Nefmut referred to as NefmutPL. The analysis of in vitro-produced EVs has supported the uploading of N protein when fused with truncated Nefmut. Mice injected with DNA vectors expressed each fusion protein developed robust SARS-CoV-2 N-specific CD8+ T cell immune responses. When ex vivo human dendritic cells were challenged with EVs engineered with either fusion products, the induction of a robust N-specific CTL activity, as evaluated by both CD107a and trogocytosis assays, was observed. Through these data we achieved the proof-of-principle that engineered EVs can be instrumental to elicit anti-SARS-CoV-2 CTL immune response in human cells. This achievement represents a mandatory step towards the upcoming experimentations in pre-clinical models focused on intranasal administration of N-engineered EVs.

1. Introduction

The systemic administration of current vaccines against respiratory viruses can result in a temporary immunity, mostly based on the production of specific antibodies [1,2]. Besides antibody waning, their major limitation consists of a fleeting to undetectable immune memory at the viral port of entry, i.e., the mucosa of high- and low-respiratory tracts, where B-memory cells were found rarely despite their presence in the blood of vaccinees [3,4,5,6]. This defect can be hardly overcome since lung cell immunity is largely not influenced by that developed in peripheral circulation and lymphoid organs. Lymphocytes in lungs are maintained independently of the pool of circulating lymphocytes, and their continuous loss through intraepithelial migration towards airways is replenished by homeostatic proliferation. This applies to CD8+ T lymphocytes [7,8] and, as demonstrated by recent findings, B lymphocytes also. On this subject, CXCR3+ CD62L− lung-resident memory B cells (BRMCs) were described as the major memory effectors of humoral antiviral immunity [9]. Based on these findings, blocking virus replication at the mucosa of upper respiratory tract represents a key step in the fight against respiratory viruses [10].
Although a robust CD8+ T-cell response is strongly beneficial in the fight against SARS-CoV-2 infection [11], no vaccine technology specifically devoted to the induction of this branch of adaptive immunity is currently available for humans. Differently to what is observed with the humoral immunity, SARS-CoV-2-specific CD8+ T cell immunity is expected to maintain intact its efficacy in the presence of the amino acid substitutions occurring in emerging viral variants [12,13]. In addition, based on data obtained with patients recovered from SARS-CoV infection, CD8+ T cell antiviral immunity is expected to last more than a decade [14].
We developed an original strategy to induce CTL immunity based on the in vivo engineering of extracellular vesicles (EVs). All cell types constitutively release EVs nanovesicles, which are key players of intercellular communication [15]. When produced by healthy cells, EVs comprise exosomes and microvesicles. Exosomes are lipid bilayer vesicles of 50–200 nm in diameter and form intracellularly upon inward invagination of endosome membranes. Intraluminal vesicles (ILVs) produced in this way form multivesicular bodies (MVBs) that can be trafficked to plasma membrane and released. Microvesicles are up to 1000 nanometers in diameter and show both physical and biochemical features similar to those of exosomes, albeit being generated through direct extrusion of plasma membrane.
We previously identified a biologically inactive Human Immunodeficiency Virus (HIV)-Type 1 Nef protein (Nefmut) having an unusually high efficiency of incorporation into EVs even when foreign polypeptides are fused to its C-terminus [16,17]. After the intramuscular (i.m) injection of DNA vectors coding for either viral or tumor antigens fused with Nefmut, high amounts of the fusion protein are uploaded in muscle cells-derived EVs and are supposed to be internalized by tissue-resident antigen-presenting cells (APCs). These cells cross-present EV contents to activate antigen-specific CD8+ T cells. In addition, these “in vivo” engineered EVs are expected to freely circulate into the body, thus having the potential to reach distal tissues.
We recently demonstrated both immunogenicity and efficacy in mouse DNA vectors expressing diverse SARS-CoV-2 structural antigens fused with Nefmut and injected intramuscularly in mice [18,19]. To validate a novel strategy of immunization at the viral port of entry based on engineered EVs, we here evaluated the CTL immunogenicity of purified EVs engineered with SARS-CoV-2 N fused with either full-length Nefmut or a C-terminal truncated form of it (referred to as NefmutPL) [20], whose characteristics could represent an improvement in terms of the safety profile of the immunogen.

2. Materials and Methods

2.1. DNA Vector Synthesis

Nefmut, Nefmut/N and NefmutPL open reading frames (ORFs) were cloned into pVAX1 plasmid (Thermo Fisher, Waltham, MA, USA), i.e., a vector approved by FDA for use in humans, as previously described [14,15,16]. ORFs coding for NefmutPL fused with SARS-CoV-2 N protein was cloned into pVAX1 plasmid as well. To this aim, an intermediate vector referred to as pVAX1/Nefmutfusion was used [18]. Here, the NefmutPL ORF deprived of its stop codon was followed by a sequence coding a GPGP linker, including a unique Apa I restriction site. In this way, sequences comprising the Apa I site at their 5′ end, and the Pme I one at their 3′ end were fused in frame with NefmutPL ORF. Stop codons was preceded by sequences coding for a DYKDDDK epitope tag (flag-tag). The vector was synthesized by Explora Biotech.

2.2. Cell Cultures and Transfection

Both human embryonic kidney (HEK) 293T cells (ATCC, CRL-11268) and human HLA-A02 MCF-7 cells (ATCC, HTB-22) were grown in DMEM (Gibco, Thermo Fisher, Waltham, MA, USA) plus 10% heat-inactivated fetal calf serum (FCS, Gibco, Thermo Fisher, Waltham, MA, USA). Transfection assays were performed using Lipofectamine 2000 (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA)-based method. Monocytes were isolated from peripheral blood mononuclear cells (PBMCs) of HLA-A.02 healthy donors by immune magnetic protocol. Human immature dendritic cells (iDCs) were obtained after 5 to 7 days of the culture of monocytes in the presence of both IL-4 and GM-CSF as described [21]. The iDCs were routinely characterized by fluorescence-activated cell sorting (FACS) analysis for the expression of CD1a, CD14, CD80, CD83, and CD86 markers. All required antibodies were from eBio, Thermo Fisher Scientific. DC maturation was obtained through overnight treatment with 10 ng/mL of lipopolysaccharide (LPS) and verified by FACS analysis.

2.3. EV Isolation

HEK293T cells transfected with vectors expressing the Nefmut-based fusion proteins were washed 24 h later and reseeded in medium supplemented with EV-deprived FCS. Supernatants were harvested from 48 to 72 h after transfection. EVs were recovered through differential centrifugations [22] by centrifuging supernatants at 500× g for 10 min, and then at 10,000× g for 30 min. Supernatants were harvested, filtered with 0.22 μm pore size filters, and ultracentrifuged at 70,000× g for l h. Pelleted vesicles were resuspended in 1×PBS, and ultracentrifuged again at 70,000× g for 1 h. Afterwards, pellets containing EVs were resuspended in 1:100 of the initial volume.

2.4. Western Blot Analysis

Western blot analyses of both cell lysates and EVs were carried out after resolving samples in 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The analysis on cell lysates was carried out by washing cells twice with 1×PBS (pH 7.4) and lysing them with 1× SDS-PAGE sample buffer. Samples were resolved by SDS-PAGE and transferred by electroblotting on a 0.45 μm pore size nitrocellulose membrane (Amersham) overnight using a Bio-Rad Trans-Blot. EVs were lysed and analyzed as described for cell lysates. For immunoassays, membranes were blocked with 5% non-fat dry milk in PBS containing 0.1% Triton X-100 for 1 h at room temperature, then incubated ON at 4 °C with specific antibodies diluted in PBS containing 0.1% Triton X-100. Filters were revealed using 1:1000-diluted sheep anti-Nef antiserum ARP 444 (a generous gift of M. Harris, University of Leeds, UK), 1:500-diluted anti-β-actin AC-74 mAb from Sigma (St. Louis, MO, USA), 1:500 diluted anti-CD81 monoclonal Ab cat. N.10616D from Thermo Fisher Scientific were analyzed by a Chemi-Doc apparatus, Bio-Rad, and relevant signals were quantified by Image Lab software version 6.1.

2.5. Mice Immunization

Six-weeks old C57 Bl/6 were obtained from Charles River. They were hosted at the Central Animal Facility of the Istituto Superiore di Sanità (ISS), as approved by the Italian Ministry of Health, authorization n. 565/2020 released on 3 June 2020. The day before the first inoculation, microchips from DATAMARS were inserted sub cute at the back of neck between the shoulder blades on the dorsal midline. Preparations of 10 μg for each DNA vector were diluted in 30 μL of sterile 0.9% saline solution. Both quality and quantity of the DNA preparations were checked by 260/280 nm absorbance and electrophoresis assays. Mice were anesthetized with isoflurane as prescribed in the Ministry authorization. Each inoculum volume was, therefore, measured by micropipette, loaded singly into a 1 mL syringe without dead volume, and injected into mouse quadriceps. Immediately after inoculation, mice underwent electroporation at the site of injection through the Agilpulse BTX device using a 4-needle array 4 mm gap, 5 mm needle length, with the following parameters: 1 pulse of 450 V for 50 µs; 0.2 ms interval; 1 pulse of 450 V for 50 µs; 50 ms interval; 8 pulses of 110 V for 10 ms with 20 ms intervals. The same procedure was repeated for both quadriceps of each mouse. Immunizations were repeated after 14 days. Fourteen days after the second immunization, mice were sacrificed by either cervical dislocation or CO2 inhalation, following the recommendations included in the Ministry authorization protocol.

2.6. Cell Isolation from Immunized Mice

Spleens were explanted by qualified personnel of ISS Central Animal Facility and transferred into a 60 mm Petri dish containing 2 mL of RPMI 1640 (Gibco, Thermo Fisher, Waltham, MA, USA), 50 µM 2-mercaptoethanol (Sigma, St. Louis, MO, USA). Splenocytes were extracted by incising the spleen with sterile scissors and pressing the cells out of the spleen sac with the plunger seal of a 1 mL syringe. After addition of 2 mL of RPMI medium, cells were transferred into a 15 mL conical tube, and the Petri plate was washed with 4 mL of medium to collect remaining cells. After a three-minute sedimentation, splenocytes were transferred to a new sterile tube to remove cell/tissue debris. Counts of live cells were carried out by the trypan blue exclusion method. Fresh splenocytes were resuspended in RPMI complete medium, containing 50 µM 2-mercaptoethanol and 10% FBS, and tested by IFN-γ EliSpot assay.

2.7. IFN-γ EliSpot Analysis

A total of 2.5 × 105 live cells were seeded in each IFN-γ EliSpot microwell. Cultures were run in triplicate in EliSpot multiwell plates (Millipore, Burlington, MA, USA, cat n. MSPS4510) pre-coated with the AN18 mAb against mouse IFN-γ (Mabtech, Nacka Strand, Sweden) in RPMI 1640 (Gibco, Thermo Fisher, Waltham, MA, USA), 10% FCS, 50 µM 2-mercaptoethanol (Sigma, St. Louis, MO, USA) for 16 h in the presence of 5 µg/mL of the CD8-specific SARS-CoV-2 N (H2-Kb) 219–228 ALALLLLDRL [23] or HIV-1 Nef (H2-Kb) 48–56 TAATNADCA peptides [24]. As negative control, 5 µg/mL of a H2-Kb-binding peptide were used. For cell activation control, cultures were treated with 10 ng/mL phorbol 12-myristate 13-acetate (PMA, Sigma) plus 500 ng/mL of ionomycin (Sigma). After 16 h, cultures were removed, and wells incubated with 100 µL of 1 µg/mL of the R4-6A2 biotinylated anti-IFN-γ (Mabtech) for 2 h at r.t. Wells were then washed and treated for 1 h at r.t. with 1:1000 diluted streptavidine-ALP preparations from Mabtech. After washing, spots were developed by adding 100 µL/well of SigmaFast BCIP/NBT, Cat. N. B5655. Spot-forming cells were finally analyzed and counted using an AELVIS EliSpot reader.

2.8. NTA Analysis of Nanovesicles

For nanoparticle tracking analysis (NTA), purified EVs were diluted in PBS and assayed with a Nanosight NS300 with the NTA software (Malvern Panalytical Ltd., Malvern, UK) through a 488 nm laser.

2.9. Cross-Priming Assay

Immature (i)DCs were challenged by engineered EVs uploading either Nefmut, Nefmut/N, or NefmutPL/N isolated from supernatants of HEK293T transfected cells. After overnight incubation, iDCs were matured by LPS treatment for 24 h. Thereafter, DCs were washed, and co-cultured with autologous peripheral blood lymphocytes (PBLs) in a 1:10 cell ratio. A week later, the stimulation procedure was repeated, and, after an additional week, CD8+ T cells were recovered for downstream assays.

2.10. Activation-Induced Degranulation and Trogocytosis Assays

Activation-induced degranulation was measured by evaluating CD107a surface expression as described [25]. Briefly, 2 × 105 PBLs recovered from cross-primed cultures were co-cultivated for 5 h with equal number of HLA-matched target cells, i.e., MCF-7 previously treated with 1 µg/mL of either SARS-CoV-2 N-specific or unrelated HLA-A.02 binding peptides, in the presence of both PE-conjugated anti-CD107a mAb H4A3 (BD-Pharmingen) and 0.7 µg/mL monensin (GolgiStop, BD, Biosciences, Franklin Lakes, NJ, USA). The gated CD8+ T cell populations were then analyzed by FACS for the detection of CD107a-related fluorescence.
For trogocytosis assay, MCF-7 cells were labeled with the fluorescent lipophilic dye CM-Dil (Molecular Probes, Invitrogen Thermo Fisher, Waltham, MA, USA) according to the manufacturer’s instructions with minor modifications. In detail, 106 target cells were resuspended in 1 mL of 1×PBS in the presence of 1 μM CM-Dil and incubated for 5 min at 37 °C followed by an additional incubation of 15 min at 4 °C. Cold RPMI medium containing 10% AB human serum was added to stop labeling (1:1 volume) for 10 min at 4 °C. Then, cells were washed three times with 1×PBS containing 1% AB human serum. Following resuspension in complete medium, labeled target cells were cultured for 4 h in the presence of 1 µg/mL of either SARS-CoV-2 N-specific or unrelated HLA-A.02 binding peptides, and finally co-cultured with primed PBLs (5 × 105 per well in 200 μL of total volume) in U-shaped 96-well plates in a 1:5 ratio. After incubation of 5 h at 37 °C, cells were washed twice in 1×PBS containing 0.5 mM EDTA to ensure cell dissociation, resuspended in 1×PBS supplemented with 0.5% bovine serum albumin, and stained with both FITC-conjugated anti-human CD3 and APCCy7-conjugated anti-human CD8 Abs (both from BD-Pharmingen) for FACS analysis. Dead cells were stained by adding Aqua LIVE/DEAD dye.
For both assays, HLA-A.02-binding SARS-CoV-2 N-specific peptides were: 159–167 LQLPQGTTL, 219–227 ALLLLDRL, 316–324 GMSRIGMEV, and 351–359 ILLNKHIDA [26].

2.11. Statistical Analysis

When appropriate, data are presented as mean + standard deviation (SD). In some instances, the Mann–Whitney U test was used. p < 0.05 was considered significant.

3. Results

3.1. EV Uploading of SARS-CoV-2 N upon Fusion with Nefmut/PL

We previously demonstrated that cells expressing SARS-CoV-2 N protein fused with full-length Nefmut release EVs uploaded with high amounts of the fusion product [18]. In the perspective of use in humans, reducing the size of the Nefmut anchoring protein would be of safety relevance. The Nefmu C-terminal truncation was already proven to not affect the EV uploading of HPV-E6,-E7 and SARS-CoV-2 S viral proteins [20]. To evaluate whether the C-terminal deletion of Nefmut affects the N-uploading efficiency in EVs, both intracellular accumulation and association with EV of NefmutPL/N were analyzed compared to that of Nefmut/N. To this aim, HEK293T cells were transiently transfected with the respective expression vectors and cell lysates analyzed 48 h thereafter. Meanwhile, EVs released on supernatants were collected 48–72 h after transfection, processed by differential centrifugations, and analyzed for the presence of Nef-related products by western blot (Figure 1 and Supplementary Figure S1). Cell-associated steady-state levels of the Nefmut-derivatives appeared similar, and both fusion products are uploaded in EVs. As indicated by the densitometry analysis, a stronger efficiency of EV uploading was observed when SARS-CoV-2 N was fused with full-length Nefmut.
We concluded that SARS-CoV-2 N can be engineered in EVs upon fusion with either Nefmut or NefmutPL.

3.2. SARS-CoV-2 N-Specific Immunogenicity Induced in Mice Injected with DNA Vectors Expressing Either Nefmut/N or NefmutPL/N

Next, we were interested in establishing whether the deletion of the EV anchoring protein affects the N-specific CD8+ T cell immunogenicity in mice. To this end, we compared the CD8+ T cell immune response induced in C57Bl/6 mice by injection of DNA vectors expressing either Nefmut/N or NefmutPL/N. The read-out consisted of γ-IFN ELISpot analyses that were carried out using the H2-Kb-restricted SARS-CoV-2 N- [23] or HIV-1 Nef-specific peptides [24]. The results indicated that both vectors were immunogenic. Consistently with the observed differences in the EV uploading efficiencies, a stronger CD8+ T cell immune response was observed in mice injected with the vector expressing full-length Nefmut (Figure 2). On the other hand, the Nef-specific CD8+ T cell immune response appeared less potent throughout.
Hence, whatever the EV-anchoring protein considered, EVs engineered with SARS-CoV-2 N elicit an N-specific CD8+ T cell immune response.

3.3. SARS-CoV-2 N Specific CTLs Are Generated upon Challenge of Human DCs with Engineered EVs

Focus of the present study was obtaining the proof-of-principle on the ability of SARS-CoV-2 N engineered EVs to induce a specific CTL activity in human cells. To this aim, PBMCs from HLA-A02 healthy donors were tested in cross-priming assays carried out by co-cultivating human PBLs with autologous DCs previously challenged with EVs engineered with SARS-CoV-2 N.
In detail, human iDCs were challenged with similar amounts, as evaluated by the EV quantification carried out through NTA assay (Figure 3), of EVs engineered by either Nefmut, Nefmut/N or NefmutPL/N.
After overnight culture in the presence of LPS, DCs (Supplementary Figure S2) were put in co-culture with autologous PBLs for a week. Afterwards, the challenge was repeated and, after an additional week, the presence of SARS-CoV-2-specific CTLs was tested in two ways, i.e., through CD107a and trogocytosis assays.
The N-specific cytolytic activity of CD8+ T lymphocytes was first assessed through FACS analysis of CD107a/LAMP-1, i.e., a well characterized cell membrane marker of CTL degranulation [25]. To this end, lymphocytes recovered after cross-priming cultures were co-cultivated for five hours at both 1:1 and 5:1 cell ratios with syngeneic cells (i.e., MCF-7) previously treated with either unrelated or N-specific peptides. We noticed an increase in CD107a expression within the CD8+ T cells from PBLs co-cultivated with DCs challenged with N-engineered EVs compared to those co-cultivated with DCs incubated with control EVs (Figure 4). The increase appeared more sustained in the case of PBLs isolated from co-cultures with iDCs challenged with EVs engineered with Nefmut/N compared to those from DCs challenged with NefmutPL/N EVs.
Antigen-specific CTLs can capture plasma membrane fragments from target cells while exerting the cytotoxic activity through a phenomenon referred to as trogocytosis [27]. The membrane capture is T-cell receptor dependent [28,29,30], epitope specific [30,31], and is exerted by lymphocyte clones endowed with the highest cytotoxic functions [32].
At the end of cross-priming cultures, PBLs were put in co-culture with peptide-treated MCF-7 cells previously labeled with CM-Dil, i.e., a red-fluorescent molecule specifically intercalating within cell membrane bilayers. After 5 h of incubation, the co-cultures were analyzed by FACS for the presence of CD8+/Dil+ T lymphocytes. As represented in Figure 5, a strong increase in red-fluorescent CD8+ T cells compared with control condition can be recognized in PBLs co-cultivated with DCs challenged with N-engineered EVs, indicating the actual presence of N-specific CTLs. Consistently with what was obtained with the CD107a assay, higher percentages of CD8+ T red-fluorescent cells were observed within PBLs co-cultivated with DCs challenged with Nefmut/N-engineered EVs compared to those co-cultivated with DCs treated with NefmutPL/N EVs.
Together, these data offer the proof-of-principle that EVs engineered with SARS-CoV-2 N antigen have the potential to elicit an antigen-specific CTL immune response in humans. This finding is of obvious relevance in view of a possible translation into the clinic of a strategy of anti-SARS-CoV-2 immunization at the viral port of entry based on Nefmut-based EVs.

4. Discussion

The aim of present investigations was achieving the proof-of–concept on CTL immunogenicity in human cells of EVs engineered with the SARS-CoV-2 N protein. Demonstrating the effectiveness of such a tool would open the way towards innovative clinic applications devoted to the induction of immunity at the port of entry of SARS-CoV-2, as well as additional respiratory viruses.
Current anti-SARS-CoV-2 vaccines have limited efficacy in inducing virus-specific CD8+ T cell immunity. In addition, in view of the minimal passage of immune cells from peripheral circulation into respiratory tissues, the limited amounts of circulating virus-specific B-memory cells present in vaccinated subjects result in a virtual absence of a suitable antiviral B-cell memory at the respiratory mucosa.
For all these reasons, a strategy focused on the induction of immunity at the oral mucosa is anticipated to result in a more effective shield against respiratory viruses. We here show that the treatment of DCs with EVs engineered with SARS-CoV-2 N leads to the formation of antiviral CTLs in human cells. As already demonstrated for a plethora of both viral and tumor antigens [17], the whole SARS-CoV-2 N protein can be engineered in EVs upon fusion of both full length and, even with a somewhat reduced efficiency, C-truncated Nefmut. Notably, reduced EV incorporation in the presence of Nefmut/PL as anchoring protein was previously not observed with other viral proteins, including both S1 and S2 from SARS-CoV-2 [19]. It is possible that in the case of SARS-CoV-2 N protein, the already documented contribution to Nef uploading in nanovesicles of the Nef C-terminal domain [33,34] critically influences the EV association the fusion product. Notably, the lower EV uploading efficiency of N when fused with NefmutPL paralleled with an apparent reduction in immunogenicity in mice, as well as in cross-priming assays carried out with human cells compared to that induced by N fused with full-length Nefmut. These pieces of experimental evidence support the idea that the efficiency of uploading in EVs as measurable “in vitro” through transfection experiments can predict the potency of the antigen-specific CD8+ T cell immune activation “in vivo”. A limitation of the present work is represented by the analysis of CTL immunogenicity that was limited to cells from HLA-A02 donors. Reproducing the investigations on cells with alternative HLAs would offer a more precise anticipation on the potential usefulness of engineered EVs as anti-SARS-CoV-2 immunogen.
Recent technologic advances allow large-scale production of EVs [35,36]. On the other hand, and as for most advanced experimental anti-Flu vaccines [37,38], the aerosol-mediated application of engineered EVs would ensure their encounter with mucosal dendritic cells (DCs) capable of sensing external stimuli and mounting appropriate local responses. These DCs can cross-present the contents of engineered EVs thereby initiating the CTL selection and activation in view of already described co-stimulatory signals provided by Nefmut [39], which are conserved in NefmutPL. Notably, the local application of engineered EVs would limit safety-based concerns in the use of EVs generated by cell lines. In sum, the potential advantages of this proposed vaccine strategy include recognition of all variants (considering the quite low mutation rate of the N gene), very few potential adverse events, immunity at the viral port of entry, and, on the basis of data published on SARS-CoV survivors [14], long lasting immunity.
Finally, the use of engineered EVs can be envisioned in combination with additional, nanoparticle-based immunogens able to induce antiviral humoral immunity and most desirably mucosal IgAs. In this way, through simple and safe aerosol administrations, a complete, as well as effective immunity at the viral port of entry could be attained. In the case of SARS-CoV-2, this strategy presents invaluable advantages compared to current vaccines.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/vaccines10071060/s1, Figure S1: Western blot analysis of transfected cells and EVs isolated from respective supernatants: raw data. Figure S2: FACS-based phenotype analysis of both iDCs and mDCs. Figure S3: Gating strategy applied on PBLs isolated from co-cultures with challenged DCs before analysis of either CD107a or CM-Dil positive cells.

Author Contributions

Conceptualization, M.F.; methodology, F.M., F.F., C.C., A.G., P.L., M.S.; investigation: F.M., C.C., A.G., M.S.; resources: M.F.; data curation: C.C., F.F.; writing—original draft preparation: M.F.; writing—review and editing: F.F.; funding acquisition: M.F. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by the institutional grant n. R220 from Istituto Superiore di Sanità, Rome, Italy.

Institutional Review Board Statement

The study was conducted according to the guidelines of the Declaration of Helsinki and approved by the Institutional Review Board of the Istituto Superiore di Sanità (protocol code n. 565/2020 PR, approved on 6 June 2020).

Informed Consent Statement

Not applicable.

Data Availability Statement

The data presented in this study are available on request from the corresponding author. The data are not publicly available due to patent application.

Acknowledgments

We thank Antonio Di Virgilio, Patrizia Cocco, Ferdinando Costa, Daniela Diamanti, Fabiola Diamanti, and Pietro Arciero, Istituto Superiore di Sanità, for technical support. We also thank Federica Magnani and Rosalba Duranti for secretarial assistance.

Conflicts of Interest

The authors declare that they have no conflict of interest.

References

  1. Lozano-Rodríguez, R.; Valentín-Quiroga, J.; Avendaño-Ortiz, J.; Martín-Quirós, A.; Pascual-Iglesias, A.; Terrón-Arcos, V.; Montalbán-Hernández, K.; Casalvilla-Dueñas, J.C.; Bergón-Gutiérrez, M.; Alcamí, J.; et al. Cellular and Humoral Functional Responses after BNT162b2 MRNA Vaccination Differ Longitudinally between Naive and Subjects Recovered from COVID-19. Cell Rep. 2022, 38, 110235. [Google Scholar] [CrossRef] [PubMed]
  2. Goel, R.R.; Painter, M.M.; Apostolidis, S.A.; Mathew, D.; Meng, W.; Rosenfeld, A.M.; Lundgreen, K.A.; Reynaldi, A.; Khoury, D.S.; Pattekar, A.; et al. MRNA Vaccines Induce Durable Immune Memory to SARS-CoV-2 and Variants of Concern. Science 2021, 374, abm0829. [Google Scholar] [CrossRef] [PubMed]
  3. Piano Mortari, E.; Russo, C.; Vinci, M.R.; Terreri, S.; Fernandez Salinas, A.; Piccioni, L.; Alteri, C.; Colagrossi, L.; Coltella, L.; Ranno, S.; et al. Highly Specific Memory B Cells Generation after the 2nd Dose of BNT162b2 Vaccine Compensate for the Decline of Serum Antibodies and Absence of Mucosal IgA. Cells 2021, 10, 2541. [Google Scholar] [CrossRef]
  4. Haralambieva, I.H.; Monroe, J.M.; Ovsyannikova, I.G.; Grill, D.E.; Poland, G.A.; Kennedy, R.B. Homologous and Variant-Specific Memory B-Cell and Antibody Responses after SARS-CoV-2 mRNA Vaccination. medRxiv 2021. [Google Scholar] [CrossRef]
  5. Goel, R.R.; Apostolidis, S.A.; Painter, M.M.; Mathew, D.; Pattekar, A.; Kuthuru, O.; Gouma, S.; Hicks, P.; Meng, W.; Rosenfeld, A.M.; et al. Distinct Antibody and Memory B Cell Responses in SARS-CoV-2 Naïve and Recovered Individuals after MRNA Vaccination. Sci. Immunol. 2021, 6, eabi6950. [Google Scholar] [CrossRef] [PubMed]
  6. Sokal, A.; Chappert, P.; Barba-Spaeth, G.; Roeser, A.; Fourati, S.; Azzaoui, I.; Vandenberghe, A.; Fernandez, I.; Meola, A.; Bouvier-Alias, M.; et al. Maturation and Persistence of the Anti-SARS-CoV-2 Memory B Cell Response. Cell 2021, 184, 1201–1213.e14. [Google Scholar] [CrossRef]
  7. Mackay, L.K.; Rahimpour, A.; Ma, J.Z.; Collins, N.; Stock, A.T.; Hafon, M.-L.; Vega-Ramos, J.; Lauzurica, P.; Mueller, S.N.; Stefanovic, T.; et al. The Developmental Pathway for CD103+CD8+ Tissue-Resident Memory T Cells of Skin. Nat. Immunol. 2013, 14, 1294–1301. [Google Scholar] [CrossRef] [PubMed]
  8. Schenkel, J.M.; Fraser, K.A.; Vezys, V.; Masopust, D. Sensing and Alarm Function of Resident Memory CD8+ T Cells. Nat. Immunol. 2013, 14, 509–513. [Google Scholar] [CrossRef] [Green Version]
  9. Allie, S.R.; Bradley, J.E.; Mudunuru, U.; Schultz, M.D.; Graf, B.A.; Lund, F.E.; Randall, T.D. The Establishment of Resident Memory B Cells in the Lung Requires Local Antigen Encounter. Nat. Immunol. 2019, 20, 97–108. [Google Scholar] [CrossRef] [PubMed]
  10. Bleier, B.S.; Ramanathan, M.; Lane, A.P. COVID-19 Vaccines May Not Prevent Nasal SARS-CoV-2 Infection and Asymptomatic Transmission. Otolaryngol. Head Neck Surg. 2021, 164, 305–307. [Google Scholar] [CrossRef]
  11. Federico, M. Virus-Induced CD8+ T-Cell Immunity and Its Exploitation to Contain the SARS-CoV-2 Pandemic. Vaccines 2021, 9, 922. [Google Scholar] [CrossRef] [PubMed]
  12. Redd, A.D.; Nardin, A.; Kared, H.; Bloch, E.M.; Pekosz, A.; Laeyendecker, O.; Abel, B.; Fehlings, M.; Quinn, T.C.; Tobian, A.A.R. CD8+ T-Cell Responses in COVID-19 Convalescent Individuals Target Conserved Epitopes From Multiple Prominent SARS-CoV-2 Circulating Variants. Open Forum Infect. Dis. 2021, 8, ofab143. [Google Scholar] [CrossRef] [PubMed]
  13. Tarke, A.; Sidney, J.; Methot, N.; Yu, E.D.; Zhang, Y.; Dan, J.M.; Goodwin, B.; Rubiro, P.; Sutherland, A.; Wang, E.; et al. Impact of SARS-CoV-2 Variants on the Total CD4+ and CD8+ T Cell Reactivity in Infected or Vaccinated Individuals. Cell Rep. Med. 2021, 2, 100355. [Google Scholar] [CrossRef]
  14. Le Bert, N.; Tan, A.T.; Kunasegaran, K.; Tham, C.Y.L.; Hafezi, M.; Chia, A.; Chng, M.H.Y.; Lin, M.; Tan, N.; Linster, M.; et al. SARS-CoV-2-Specific T Cell Immunity in Cases of COVID-19 and SARS, and Uninfected Controls. Nature 2020, 584, 457–462. [Google Scholar] [CrossRef]
  15. Van Niel, G.; D’Angelo, G.; Raposo, G. Shdding Light on the Cell Biology of Extracellular Vesicles. Nat. Rev. Mol. Cell Biol. 2018, 19, 213–228. [Google Scholar] [CrossRef]
  16. Di Bonito, P.; Chiozzini, C.; Arenaccio, C.; Anticoli, S.; Manfredi, F.; Olivetta, E.; Ferrantelli, F.; Falcone, E.; Ruggieri, A.; Federico, M. Antitumor HPV E7-Specific CTL Activity Elicited by in Vivo Engineered Exosomes Produced through DNA Inoculation. Int. J. Nanomed. 2017, 12, 4579–4591. [Google Scholar] [CrossRef] [Green Version]
  17. Anticoli, S.; Manfredi, F.; Chiozzini, C.; Arenaccio, C.; Olivetta, E.; Ferrantelli, F.; Capocefalo, A.; Falcone, E.; Ruggieri, A.; Federico, M. An Exosome-Based Vaccine Platform Imparts Cytotoxic T Lymphocyte Immunity Against Viral Antigens. Biotechnol. J. 2018, 13, e1700443. [Google Scholar] [CrossRef]
  18. Ferrantelli, F.; Chiozzini, C.; Manfredi, F.; Giovannelli, A.; Leone, P.; Federico, M. Simultaneous CD8 + T-Cell Immune Response against SARS-CoV-2 S, M, and N Induced by Endogenously Engineered Extracellular Vesicles in Both Spleen and Lungs. Vaccines 2021, 9, 240. [Google Scholar] [CrossRef]
  19. Ferrantelli, F.; Chiozzini, C.; Manfredi, F.; Leone, P.; Spada, M.; Di Virgilio, A.; Giovannelli, A.; Sanchez, M.; Cara, A.; Michelini, Z.; et al. Strong SARS-CoV-2 N-Specific CD8+ T Immunity Induced by Engineered Extracellular Vesicles Associates with Protection from Lethal Infection in Mice. Viruses 2022, 14, 329. [Google Scholar] [CrossRef]
  20. Chiozzini, C.; Manfredi, F.; Ferrantelli, F.; Leone, P.; Giovannelli, A.; Olivetta, E.; Federico, M. The C-Terminal Domain of Nefmut Is Dispensable for the CD8+ T Cell Immunogenicity of In Vivo Engineered Extracellular Vesicles. Vaccines 2021, 9, 373. [Google Scholar] [CrossRef]
  21. Anticoli, S.; Aricò, E.; Arenaccio, C.; Manfredi, F.; Chiozzini, C.; Olivetta, E.; Ferrantelli, F.; Lattanzi, L.; D’Urso, M.T.; Proietti, E.; et al. Engineered Exosomes Emerging from Muscle Cells Break Immune Tolerance to HER2 in Transgenic Mice and Induce Antigen-Specific CTLs upon Challenge by Human Dendritic Cells. J. Mol. Med. 2018, 96, 211–221. [Google Scholar] [CrossRef] [PubMed]
  22. Théry, C.; Amigorena, S.; Raposo, G.; Clayton, A. Isolation and Characterization of Exosomes from Cell Culture Supernatants and Biological Fluids. Curr. Protoc. Cell Biol. 2006, 30, 3.22.1–3.22.29. [Google Scholar] [CrossRef] [PubMed]
  23. Zhao, J.; Zhao, J.; Perlman, S. T Cell Responses Are Required for Protection from Clinical Disease and for Virus Clearance in Severe Acute Respiratory Syndrome Coronavirus-Infected Mice. J. Virol. 2010, 84, 9318–9325. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  24. Liang, X.; Fu, T.-M.; Xie, H.; Emini, E.A.; Shiver, J.W. Development of HIV-1 Nef Vaccine Components: Immunogenicity Study of Nef Mutants Lacking Myristoylation and Dileucine Motif in Mice. Vaccine 2002, 20, 3413–3421. [Google Scholar] [CrossRef]
  25. Betts, M.R.; Koup, R.A. Detection of T-Cell Degranulation: CD107a and b. Methods Cell Biol. 2004, 75, 497–512. [Google Scholar] [CrossRef]
  26. Ahmed, S.F.; Quadeer, A.A.; McKay, M.R. Preliminary Identification of Potential Vaccine Targets for the COVID-19 Coronavirus (SARS-CoV-2) Based on SARS-CoV Immunological Studies. Viruses 2020, 12, 254. [Google Scholar] [CrossRef] [Green Version]
  27. Hudrisier, D.; Riond, J.; Mazarguil, H.; Gairin, J.E.; Joly, E. Cutting Edge: CTLs Rapidly Capture Membrane Fragments from Target Cells in a TCR Signaling-Dependent Manner. J. Immunol. 2001, 166, 3645–3649. [Google Scholar] [CrossRef] [Green Version]
  28. Stinchcombe, J.C.; Bossi, G.; Booth, S.; Griffiths, G.M. The Immunological Synapse of CTL Contains a Secretory Domain and Membrane Bridges. Immunity 2001, 15, 751–761. [Google Scholar] [CrossRef] [Green Version]
  29. Joly, E.; Hudrisier, D. What Is Trogocytosis and What Is Its Purpose? Nat. Immunol. 2003, 4, 815. [Google Scholar] [CrossRef]
  30. Beadling, C.; Slifka, M.K. Quantifying Viable Virus-Specific T Cells without a Priori Knowledge of Fine Epitope Specificity. Nat. Med. 2006, 12, 1208–1212. [Google Scholar] [CrossRef]
  31. Tomaru, U.; Yamano, Y.; Nagai, M.; Maric, D.; Kaumaya, P.T.P.; Biddison, W.; Jacobson, S. Detection of Virus-Specific T Cells and CD8+ T-Cell Epitopes by Acquisition of Peptide-HLA-GFP Complexes: Analysis of T-Cell Phenotype and Function in Chronic Viral Infections. Nat. Med. 2003, 9, 469–476. [Google Scholar] [CrossRef] [PubMed]
  32. Machlenkin, A.; Uzana, R.; Frankenburg, S.; Eisenberg, G.; Eisenbach, L.; Pitcovski, J.; Gorodetsky, R.; Nissan, A.; Peretz, T.; Lotem, M. Capture of Tumor Cell Membranes by Trogocytosis Facilitates Detection and Isolation of Tumor-Specific Functional CTLs. Cancer Res. 2008, 68, 2006–2013. [Google Scholar] [CrossRef] [Green Version]
  33. Zheng, Y.-H.; Plemenitas, A.; Fielding, C.J.; Peterlin, B.M. Nef Increases the Synthesis of and Transports Cholesterol to Lipid Rafts and HIV-1 Progeny Virions. Proc. Natl. Acad. Sci. USA 2003, 100, 8460–8465. [Google Scholar] [CrossRef] [Green Version]
  34. Cai, C.Y.; Zhang, X.; Sinko, P.J.; Burakoff, S.J.; Jin, Y.-J. Two Sorting Motifs, a Ubiquitination Motif and a Tyrosine Motif, Are Involved in HIV-1 and Simian Immunodeficiency Virus Nef-Mediated Receptor Endocytosis. J. Immunol. 2011, 186, 5807–5814. [Google Scholar] [CrossRef] [Green Version]
  35. McNamara, R.P.; Caro-Vegas, C.P.; Costantini, L.M.; Landis, J.T.; Griffith, J.D.; Damania, B.A.; Dittmer, D.P. Large-Scale, Cross-Flow Based Isolation of Highly Pure and Endocytosis-Competent Extracellular Vesicles. J. Extracell. Vesicles 2018, 7, 1541396. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  36. Paganini, C.; Capasso Palmiero, U.; Pocsfalvi, G.; Touzet, N.; Bongiovanni, A.; Arosio, P. Scalable Production and Isolation of Extracellular Vesicles: Available Sources and Lessons from Current Industrial Bioprocesses. Biotechnol. J. 2019, 14, e1800528. [Google Scholar] [CrossRef] [PubMed]
  37. Murugappan, S.; Frijlink, H.W.; Petrovsky, N.; Hinrichs, W.L.J. Enhanced Pulmonary Immunization with Aerosolized Inactivated Influenza Vaccine Containing Delta Inulin Adjuvant. Eur. J. Pharm. Sci. 2015, 66, 118–122. [Google Scholar] [CrossRef]
  38. Xu, J.; Li, S.; Wang, X.; Liu, J.; Shan, P.; Zhou, Y.; Zhao, J.; Wang, Z.; Xu, C.; Chen, M.; et al. Systemic and Mucosal Humoral Immune Responses Induced by the JY-Adjuvanted Nasal Spray H7N9 Vaccine in Mice. Emerg. Microbes Infect. 2018, 7, 140. [Google Scholar] [CrossRef] [Green Version]
  39. Sistigu, A.; Bracci, L.; Valentini, M.; Proietti, E.; Bona, R.; Negri, D.R.M.; Ciccaglione, A.R.; Tritarelli, E.; Nisini, R.; Equestre, M.; et al. Strong CD8+ T Cell Antigenicity and Immunogenicity of Large Foreign Proteins Incorporated in HIV-1 VLPs Able to Induce a Nef-Dependent Activation/Maturation of Dendritic Cells. Vaccine 2011, 29, 3465–3475. [Google Scholar] [CrossRef]
Vaccines 10 01060 g001 550
Figure 1. Detection of Nefmut-related products in cells and EVs after DNA transfection. Western blot analysis on 30 μg of lysates from HEK293T cells transfected with DNA vectors expressing either Nefmut/N, NefmutPL/N, or, as control, void pVAX1 vector (Ctrl) (left panels). Equal volumes of buffer where purified EVs were resuspended after differential centrifugations of the respective cell supernatants were also analyzed (right panels). Polyclonal anti-Nef Abs served to detect Nefmut-based products, while β-actin and CD81 were revealed as markers for cell lysates and EVs, respectively. Molecular markers are given in kilodaltons (kDa). Arrows sign relevant products. Intensity ratios between NefmutPL/N and Nefmut/N signals as revealed by densitometry analysis are also indicated. The results are representative of three independent experiments.
Figure 1. Detection of Nefmut-related products in cells and EVs after DNA transfection. Western blot analysis on 30 μg of lysates from HEK293T cells transfected with DNA vectors expressing either Nefmut/N, NefmutPL/N, or, as control, void pVAX1 vector (Ctrl) (left panels). Equal volumes of buffer where purified EVs were resuspended after differential centrifugations of the respective cell supernatants were also analyzed (right panels). Polyclonal anti-Nef Abs served to detect Nefmut-based products, while β-actin and CD81 were revealed as markers for cell lysates and EVs, respectively. Molecular markers are given in kilodaltons (kDa). Arrows sign relevant products. Intensity ratios between NefmutPL/N and Nefmut/N signals as revealed by densitometry analysis are also indicated. The results are representative of three independent experiments.
Vaccines 10 01060 g001
Vaccines 10 01060 g002 550
Figure 2. SARS-CoV-2-specific CD8+ T cell immunity induced in DNA injected mice. CD8+ T cell immune response in C57 Bl/6 mice inoculated i.m. with DNA vectors expressing either Nefmut/N, NefmutPL/N or, as control, pVAX1. At the time of sacrifice, 2.5 × 105 splenocytes were incubated ON with or without 5 µg/mL of either unrelated, SARS-CoV-2-N, or HIV-1 Nef-specific peptides in replicate IFN-γ EliSpot microwells. In panel (A), the raw data obtained with splenocytes from mice injected with either Nefmut/N or NefmutPL/N are reported. PMA indicates cell cultures treated with PMA plus ionomycin. In panel (B), shown are the numbers of IFN- γ SFUs/well as mean values of triplicates after subtraction of mean values measured in wells seeded with splenocytes treated with unspecific peptides. Intragroup mean values + SD are also shown. The results are representative of two independent experiments. * p < 0.05.
Figure 2. SARS-CoV-2-specific CD8+ T cell immunity induced in DNA injected mice. CD8+ T cell immune response in C57 Bl/6 mice inoculated i.m. with DNA vectors expressing either Nefmut/N, NefmutPL/N or, as control, pVAX1. At the time of sacrifice, 2.5 × 105 splenocytes were incubated ON with or without 5 µg/mL of either unrelated, SARS-CoV-2-N, or HIV-1 Nef-specific peptides in replicate IFN-γ EliSpot microwells. In panel (A), the raw data obtained with splenocytes from mice injected with either Nefmut/N or NefmutPL/N are reported. PMA indicates cell cultures treated with PMA plus ionomycin. In panel (B), shown are the numbers of IFN- γ SFUs/well as mean values of triplicates after subtraction of mean values measured in wells seeded with splenocytes treated with unspecific peptides. Intragroup mean values + SD are also shown. The results are representative of two independent experiments. * p < 0.05.
Vaccines 10 01060 g002
Vaccines 10 01060 g003 550
Figure 3. NTA analysis of EVs from transfected HEK 293T cells. EVs from cells transfected with either Nefmut/N, NefmutPL/N or, as control, pVAX1 (Ctrl) were isolated by differential centrifugation, and resuspended in the same volumes of buffer. Preparations were then diluted 20 times and analyzed 5 times with a Nanosight NS300 device. Size distribution and average concentration of EVs are reported as calculated. The results are representative of two independent experiments.
Figure 3. NTA analysis of EVs from transfected HEK 293T cells. EVs from cells transfected with either Nefmut/N, NefmutPL/N or, as control, pVAX1 (Ctrl) were isolated by differential centrifugation, and resuspended in the same volumes of buffer. Preparations were then diluted 20 times and analyzed 5 times with a Nanosight NS300 device. Size distribution and average concentration of EVs are reported as calculated. The results are representative of two independent experiments.
Vaccines 10 01060 g003
Vaccines 10 01060 g004 550
Figure 4. CTL activity as revealed in terms of CD107a expression in CD8+ T lymphocytes co-cultivated with DCs challenged with engineered EVs. CD107a FACS analysis on HLA-A.02 PBLs recovered after cross-priming with DCs challenged with EVs isolated from supernatant of cells transfected with pVAX1 (Ctrl), Nefmut/N, NefmutPL/N. PBLs were analyzed 5 h after co-culture with HLA-A.02 MCF-7 cells pre-treated with either the unrelated or the SARS-CoV-2-specific peptide. The results in panel (A) are representative of five independent experiments carried out with cells from different HLA-A.02 healthy donors. Full data (1:1 cell ratio) are plotted in panel (B). Gating strategy (Supplementary Figure S3): co-cultures labeled with both FITC-conjugated anti-CD3 and APCCy7-conjugated anti-CD8 mAbs were selected for CD3 expression (for PBL identification), and then the CD8+ cell sub-populations were identified within CD3+ cells. Quadrants were set based on the fluorescence detected after cell labeling with relevant IgG isotypes. * p < 0.05.
Figure 4. CTL activity as revealed in terms of CD107a expression in CD8+ T lymphocytes co-cultivated with DCs challenged with engineered EVs. CD107a FACS analysis on HLA-A.02 PBLs recovered after cross-priming with DCs challenged with EVs isolated from supernatant of cells transfected with pVAX1 (Ctrl), Nefmut/N, NefmutPL/N. PBLs were analyzed 5 h after co-culture with HLA-A.02 MCF-7 cells pre-treated with either the unrelated or the SARS-CoV-2-specific peptide. The results in panel (A) are representative of five independent experiments carried out with cells from different HLA-A.02 healthy donors. Full data (1:1 cell ratio) are plotted in panel (B). Gating strategy (Supplementary Figure S3): co-cultures labeled with both FITC-conjugated anti-CD3 and APCCy7-conjugated anti-CD8 mAbs were selected for CD3 expression (for PBL identification), and then the CD8+ cell sub-populations were identified within CD3+ cells. Quadrants were set based on the fluorescence detected after cell labeling with relevant IgG isotypes. * p < 0.05.
Vaccines 10 01060 g004
Vaccines 10 01060 g005 550
Figure 5. Trogocytosis analysis carried out with CD8+ T lymphocytes recovered after cross-priming co-cultures. FACS analysis of PBLs recovered, as described in Figure 4, and co-cultivated with N-peptides treated, CM-Dil-labeled MCF-7 cells. Gating strategy: co-cultures labeled with both FITC-conjugated anti-CD3 and APCCy7-conjugated anti-CD8 mAbs were selected for CD3 expression (for PBL identification), and then the CD8+ cells were identified within CD3+ cell populations. CD8-labeled cells were finally scored for the CM-Dil-specific fluorescence. Quadrants were set based on the background fluorescence as detected after cell labeling with relevant IgG isotypes. Data are representative of two independent experiments carried out with cells from different HLA-A02 healthy donors.
Figure 5. Trogocytosis analysis carried out with CD8+ T lymphocytes recovered after cross-priming co-cultures. FACS analysis of PBLs recovered, as described in Figure 4, and co-cultivated with N-peptides treated, CM-Dil-labeled MCF-7 cells. Gating strategy: co-cultures labeled with both FITC-conjugated anti-CD3 and APCCy7-conjugated anti-CD8 mAbs were selected for CD3 expression (for PBL identification), and then the CD8+ cells were identified within CD3+ cell populations. CD8-labeled cells were finally scored for the CM-Dil-specific fluorescence. Quadrants were set based on the background fluorescence as detected after cell labeling with relevant IgG isotypes. Data are representative of two independent experiments carried out with cells from different HLA-A02 healthy donors.
Vaccines 10 01060 g005
Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Share and Cite

MDPI and ACS Style

Manfredi, F.; Chiozzini, C.; Ferrantelli, F.; Leone, P.; Giovannelli, A.; Sanchez, M.; Federico, M. Activation of Anti-SARS-CoV-2 Human CTLs by Extracellular Vesicles Engineered with the N Viral Protein. Vaccines 2022, 10, 1060. https://doi.org/10.3390/vaccines10071060

AMA Style

Manfredi F, Chiozzini C, Ferrantelli F, Leone P, Giovannelli A, Sanchez M, Federico M. Activation of Anti-SARS-CoV-2 Human CTLs by Extracellular Vesicles Engineered with the N Viral Protein. Vaccines. 2022; 10(7):1060. https://doi.org/10.3390/vaccines10071060

Chicago/Turabian Style

Manfredi, Francesco, Chiara Chiozzini, Flavia Ferrantelli, Patrizia Leone, Andrea Giovannelli, Massimo Sanchez, and Maurizio Federico. 2022. "Activation of Anti-SARS-CoV-2 Human CTLs by Extracellular Vesicles Engineered with the N Viral Protein" Vaccines 10, no. 7: 1060. https://doi.org/10.3390/vaccines10071060

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop