T cell immunity of the nonadjuvanted HLA-restricted peptide COVID-19 vaccine

doi:10.21203/rs.3.rs-2119898/v1

Download PDF

Article

T cell immunity of the nonadjuvanted HLA-restricted peptide COVID-19 vaccine

https://doi.org/10.21203/rs.3.rs-2119898/v1

This work is licensed under a CC BY 4.0 License

Version 1

posted

You are reading this latest preprint version

Recently, the cases of breakthrough infection and restored virus of COVID-19 have increased after full vaccination, which might be contributed by immune surveillance escape or rebound virus. Here, artificial linear 9-mer human leucocyte antigen (HLA)-restricted UC peptides are designed based on the well-conserved S2 region of the COVID-19 spike protein regardless of rapid mutation and glycosylation hindrance. Through HLA molecule presentation, UC peptides can activate cytotoxic T lymphocytes (CTLs), which elicit cytotoxic activity by recognizing COVID-19 spike-bearing cells and preferably secreting Th1 cytokines. The UC peptides showed immunogenicity and generated a specific antibody in mice either by intramuscular injection or oral delivery without an adjuvant formulation. In conclusion, the T cell vaccine could provide long-lasting protection against COVID-19 either during reinfection or during the rebound of COVID-19. With the eradication of COVID-19 virus-infected cells, the COVID-19 T cell vaccine might provide a solution to lower COVID-19 severity and long COVID.

Biological sciences/Immunology/Vaccines

Health sciences/Diseases/Infectious diseases/Viral infection

The rapid transmission of SARS-CoV-2 since the 2019 December initial outbreak has caused more than 608 million infections and 6.5 million deaths in September 2022 (https://covid19.who.int/). However, it continued to spread because of the nature of an RNA virus that is prone to mutation. To date, the COVID-19 virus has evolved several variants of concern (VOCs) since alpha, omicron and omicron subvariants such as BA2.75, BA.4 and BA.5. Currently, many COVID-19 vaccines have been approved to vaccinate people; however, COVID-19 viruses are still evolving¹. SARS-CoV-2 becomes more contagious and less lethal, but it still affects the vulnerable population to develop multi-inflammatory syndrome (MIS)^2,3.

To prevent COVID-19 infection, vaccination is an important strategy to generate neutralizing antibodies to stop COVID-19 spread. In the emergence of the COVID-19 pandemic, the spike protein was widely developed as a vaccine antigen target, especially in the receptor-binding domain (RBD), for antiviral infection. However, the spike is the rapidly mutated SARS-CoV-2 virus protein, whose mutation sites frequently occur on the RBD of SARS-CoV-2. Although the original spike antigen vaccine can successfully be effective against wild-type SARS-CoV-2, the subsequent evolution of SARS-CoV-2 variants, such as alpha, beta, gamma, delta and omicron, was reported to cause gradual loss of virus neutralization protection. Their virus neutralization titer was dramatically reduced 21-fold compared to the wild type by BA.4/BA.5 observed in people after booster vaccination⁴. Breakthrough infection was frequently diagnosed after a new COVID-19 variant occurred, which may be contributed by the low neutralizing antibody level or loss of cross-binding capability to new COVID-19 variants⁵. Currently, the omicron subvariants BA.4 and BA.5 are the dominant cocurrent circulating VOCs under monitoring by the World Health Organization (WHO, https://www.who.int/activities/tracking-SARS-CoV-2-variants).

To date, more than 4.9 billion people have been fully vaccinated. At least 2.4 billion people have received booster vaccines to enhance protection against new VOC subvariants, such as BA.4, BA.5 and BA2.75 (https://ourworldindata.org/covid-vaccinations). This may raise the question of whether current vaccines cannot protect against all COVID-19 variants and urgently need a new strategy to develop an effective vaccine to generate broadly neutralizing antibodies and long-term memory cellular immunity against COVID-19 variants and subvariants^6,7.

To date, we have already experienced 5 VOCs of SARS-CoV-2 within 2 years despite the 60 to 80% vaccination coverage rate in developing or developed countries. The goal of herd immunity is still far from achievement because of unstable SARS-CoV-2 that accumulates massive mutations, such as Omicron BA.1, which has 39 mutations⁸. Furthermore, a new VOC of the current COVID-19 pandemic appeared approximately every 6 months, as reported by the WHO. However, none of the effective vaccines can catch out on time because they require the completed vaccine development process, including the design, proof, validation, manufacturing, safety evaluation and regulatory approval. Therefore, a wide protection of universal vaccines against all kinds of variants is an urgent need for controlling the COVID-19 pandemic⁹.

Compared to humoral immunity, cellular immunity contributes to long-lasting immunity, in which specific memory T cells can be detected after 10 years, whereas specific antibodies decrease to low neutralizing levels in 2–3 months and undetectable levels within 2–3 years^10,11. It might be proposed that cellular immunity is critical for preventing COVID-19 infection rather than humoral immunity, especially in COVID-19 vaccine cases¹². Many studies and surveys have been published to support that the cellular immunity vaccine against COVID-19 is more effective than humoral immunity at the stage of initial virus infection and long COVID¹³. In our study, we demonstrated an HLA-restricted peptide vaccine derived from the well-conserved region of the S2 domain of the COVID-19 spike protein. Cellular and humoral immunity were determined in vitro and in vivo. Oral availability was also demonstrated to support gut immunity. It is expected that both humoral and cellular immunity will develop to fully protect against COVID-19 infection.

UC peptides designed from the well-conserved region of the COVID-19 spike protein for HLA restriction.

The epitopes of UC-100 and UC-152 were designed from the S2 domain of the COVID-19 virus spike protein, in which the well-conserved region was selected according to the side chain of a specific amino acid based on steric protein structure (Fig. 1a). The UC peptides can be directly assigned to avoid virus mutations and glycosylation sites within this well-conserved region. The side chain of each amino acid was aligned for a structural linear sequence according to the distance of approximately 3.8 Å of Cα-Cα between 2 amino acids on the protein surface. Artificial UC-100 peptides comprising 9 amino acids were designed and expected to bind with the major histocompatibility complex (MHC)-class I molecule. The binding affinity to various MHC-I molecules was predicted, as shown in Table 1. The artificial UC-100 antigen was predicted to not only strongly bind to HLA-A201 but also bind to other major histocompatibility molecules. The artificial UC-100 epitope antigen sequence aligned on the surface of the COVID-19 spike protein structure, which was discontinuous in the primary sequence and assembled into a linear arrangement on the surface (Fig. 1b). The discontinued sequence could not be processed as known immunological antigen presenting on the immune cells, but the surface antigenicity might provoke cellular immunity. Due to spike polypeptide refolding, the discontinued primary sequence of the spike protein formed a linear alignment on the surface. To obtain another higher binding activity of antigen peptides to MHC molecules, the artificial UC-152 was modified with one amino acid substitute of the UC-100 peptide. The UC-152 peptide showed a stronger binding affinity to various MHC-I molecules, as shown in Table 1. Both UC-100 and UC-152 peptides were designed to boost cellular immunity.

Table 1

The prediction of UC peptides binding to various major histocompatibility variants
	HLA-A/ IC₅₀ (nM)		HLA-B/ IC₅₀ (nM)		HLA-C/ IC₅₀ (nM)
UC-100	02:01 02:06 03:01 30:01 30:02 31:01 32:01	167.9 267.6 6711.2 2420.9 998.8 5134.2 4139.4	15:02	1303.9	03:03 05:01 07:01 08:02 12:03 14:02 15:02	3339.0 46.0 7398.9 8397.7 28.9 148.5 1053.0
UC-152	02:01 02:06 03:01 30:01 30:02 31:01 32:01	51.4 79.0 2311.0 1577.5 838.4 2821.4 2985.0	15:02	1880.3	03:03 05:01 07:01 08:02 12:03 14:02 15:02	7561.5 82.0 3708.3 5796.4 28.9 148.5 938.5
The predicted binding affinity of peptide to MHC-I was analyzed with SMM on the Immune Epitope Database website (http://www.iedb.org/)

The UC peptides could be primed onto the CTLs and engaged with the corresponding CTLs

To evaluate the binding affinity of UC-100 and UC-152 to MHC-I molecules, the HLA-A201 complex was analyzed in an HLA refolding assay. The anti-β₂m complex antibody can recognize the correct folded structure when the matched peptide is loaded on the MHC-I molecule along with β₂m. In the refolding assay, both UC-100 and UC-152 showed a higher binding activity to HLA-A201 in a dose-dependent manner than the cytomegalovirus (CMV)-positive antigen (Fig. 2a). The results showed that both the 9-mer UC-100 and UC-152 peptides could refold with the HLA-A201 molecule and β₂m. It was indicated that the artificial design of linear-aligned antigens UC-100 and UC-152 could be loaded on the HLA class I molecule and might be recognized by corresponding CTLs. To verify whether corresponding CTLs existed, the UC-100 and UC-152 antigens were subjected to the tetramer assay, and the corresponding CTLs were stained with the labeled tetramer complex. In the tetramer assay, HLA-A201 molecules refolded with UC-100 and UC-152 showed increases of 2.2% and 3.3% positive CTLs compared with tetramers without loading peptide (Fig. 2b). Those correctly refolded complexes would be engaged with the corresponding T cell receptor in CD8⁺-CTLs in human peripheral blood mononuclear cells (PBMCs). These results showed that both UC-100 and UC-152 peptides could form a complex with HLA and trigger the activation of CTLs through the MHC/TCR complex.

UC peptides could be presented to MHC class II molecules and interact with T helper cells

Although the HLA-DR molecule can be loaded up to 15-mer antigenic peptide in length, it can present loaded antigen to the responsible T helper cells. Thus, the 9-mer UC peptides were evaluated for the engagement of T-helper cells with the formation of the HLA-DR/peptide complex. To verify the role of antigen peptides involved in the interaction between HLA-DR and TCRs, the peptides were subjected to the dextramer assay to understand the engagement of human T helper cells. Human PBMCs were used for the dextramer assay, and the mean fluorescence intensity (MFI) of the labeled dextramer was analyzed to determine the level of engaged CD4⁺-positive cells. In the dextramer assay, the results showed that UC-100 and UC-152 peptides could form a complex with HLA-DR and CD4⁺-T helper cells that were stained to increase 3.1-fold and 2.7-fold compared to the non-peptide loading group, respectively (Fig. 2c). It was indicated that 9-mer UC-100 and UC-152 peptides could form the HLA DR/peptide complex and interact with T helper cells via HLA DR/TCR engagement. This finding suggests that 9-mer UC peptides might also be presented to immune cells via HLA DR/TCR molecules.

UC peptides polarized the Th1 cytokine release of human PBMCs

Immune cells, such as T helper cells, can secrete cytokines to support the immune response toward Th1 (IFN-γ) or Th2 (IL-4). To understand the cytokine release pattern of the immune response in response to UC peptides, human PBMCs were treated with different concentrations of UC-100 and UC152 peptides. The intracellular cytokines IFN-γ and IL-4 of T helper cells were assayed to represent the Th1 and Th2 responses. The results showed that both UC-100 and UC-152 preferably stimulated IFN-γ cytokine secretion rather than IL-4 cytokine secretion (Fig. 2d, 2e). The level of IFN-γ was elevated in response to both UC-100 and UC-152 in a dose-dependent manner. This meant that those 9-mer UC peptides preferably support the Th1 response.

UC peptides could directly activate the specific lysis activity of CTLs

According to the previous results, UC peptides could form the MHC-I/peptide complex, and a number of CTLs were detected to be engaged. It might directly activate the CTLs to attack the antigen-bearing cells. To verify the effect of peptide-primed immune cells, the lysis activity of CTLs was assessed by targeting spike-expressing cells. In the CTL lysis assay, spike-expressing cells cocultured with different ratios of PBMCs were treated with UC-100 peptides at 9 nM and 27 nM. The results showed that the UC-100 peptide increased the lysis activity of spike-expressing cells in a dose-dependent manner (Fig. 3a). Similar results were observed in UC-152 peptide-treated experiments (Fig. 3b). These results supported that spike-expressing cells could be recognized and were cytotoxic by CTLs, which CTLs were educated by UC peptides. It was also demonstrated that UC peptides could activate immune cells to lyse virus-infected cells.

The nonadjuvanted UC peptides provoked immunogenicity in vivo.

The mice were immunized by intramuscular injection with UC-100 and UC-152 without adjuvant formulation. The total IgG, anti-peptide (UC-100 or UC-152)-specific IgG and anti-spike trimer were assayed for the immunogenicity of UC peptides. The results showed that total mouse IgG in postdose serum could be induced by UC-100 peptide to 339 µg/mL and by UC-152 to 340 µg/mL compared to 186 µg/mL and 127 µg/mL IgG in the predose mouse background (Fig. 4a, 4d). There was a 1.8 ~ 2.7-fold increase in IgG production with UC peptide challenge. The immunized serum was also subjected to a specific binding assay to the UC-100 and UC-152 peptide-coated microplates. The results showed that approximately 1.6-fold and 1.8-fold of serum antibody were significantly increased to bind to corresponding coated UC-100 and UC-152 peptides (Fig. 4b, 4e). This result indicated that antibodies induced by UC-100 and UC-152 peptides can specifically recognize antigenic peptide sequences. The results also showed that both antibodies induced by UC-100 and UC-152 peptides could also bind to the COVID-19 spike trimer protein, similar to the peptide binding results (Fig. 4c, 4f). Approximately 4.0 ~ 4.5-fold of specific anti-spike trimer IgG was increased. It was shown that 9-mer UC peptides not only showed immunogenicity in animals but also induced antibodies to recognize the matched synthetic peptide and structural sequence of the spike protein on the surface.

The UC peptide-induced antibody could activate complement to lyse epitope-bearing cells

To verify the complement effects of the vaccinated serum antibodies on virus-infected cells, the serum was tested for the complement-dependent cytotoxicity (CDC) assay in spike-expressing cells. In the CDC assay, spike-expressing HEK293 cells cultured in serum-free medium were treated with serially diluted serum supplemented with baby rabbit complements. Complement cytotoxicity was measured by lactate dehydrogenase (LDH) activity. The results showed that the complement-induced activity was increased approximately 2.1-fold in vaccinated UC-152 peptide serum compared to the pre-vaccinated serum background but not in UC-100 peptide-vaccinated mice (Fig. 4g, 4h). This result indicated that the UC peptide-induced antibody might target the intact spike protein of infected cells depending on the binding affinity of the induced antibody.

The oral administration of UC peptides provokes immunogenicity in mice

Due to digestion resistance of artificial UC peptides, immunogenicity by oral administration was evaluated to understand whether the peptide could stimulate gut immunity. The five BALB/c mice were orally administered 200 µg of polymer-formulated UC-100. After 3 doses were administered 2 weeks apart, pre-dose and post-dose serum were assayed for the levels of total IgG, specific anti-peptide and anti-COVID-19 spike IgG. The results showed that total IgG was induced by oral UC-100 peptide from 136 µg/mL to 388 µg/mL (Fig. 5a). For the specificity of the induced antibody, an approximately 1.6-fold increase in anti-peptide IgG and a 4.4-fold increase in anti-spike IgG were measured (Fig. 5b, 5c). These results suggested that oral administration of UC-100 peptide could induce an immune response through gut immunity. To understand the CDC function of the induced antibody, serum was tested for complement activity, and the results showed that antibody-mediated complement activity was slightly increased by approximately 27% in vaccinated serum compared with the pre-vaccinated serum background (Fig. 5d). These results indicated that complement activity mediated by antibodies might be involved in the eradication of virus-infected cells. To further understand the possibility of mucosa immunity from an HLA-restricted peptide vaccine, the sera were used to determine the levels of total IgA and specific IgA. The results showed that total IgA was significantly induced by oral UC-100 peptide from 13.50 µg/mL to 33.04 µg/mL, which was an approximately 2.4-fold increase in IgA (Fig. 5e). For the specificity of the induced antibody, there was an approximately 1.8-fold increase in the specific anti-peptide IgA (Fig. 5f). These results suggested that oral administration of short UC peptides could induce mucosal immunity.

In this paper, artificial 9-mer UC peptides are designed to enhance T cell immunity. We successfully demonstrated the antigenicity of these UC peptides and the possible mechanism of UC peptides in T cell and B cell activation. These data fully support the development of a 9-mer peptide as a T cell vaccine accompanying the B cell response (Fig. 6). This explains how the 9-mer short peptide activates CTLs through MHC-I recognition and activates B cells to produce antibodies via MHC-DR engagement. Based on these observations, the 9-mer HLA-restricted peptide has the potential to be used to develop a next-generation vaccine against COVID-19. To date, the COVID-19 pandemic is still threatening the world due to several VOC waves causing breakthrough infections, including Omicron and subvariants. These pandemic variants escape from human immunity and exist even in fully vaccinated or COVID-19 convalescent human serum¹⁴. There is still an urgent need to develop an effective COVID-19 vaccine to stop this pandemic. In fact, breakthrough infections with new VOCs frequently occur in nations or areas with high vaccination coverage rates¹⁵. Restored COVID-19 viruses were observed in approximately 10% of convalescent patients¹⁶. It is implied that COVID-19 viruses are not eradicated and persistently exist in a latent phase. It is deduced that there is a balance in viral load between the function of antibody-producing B cells and virus-infected cells. When the balance is collapsed, the viruses will rebound and become contagious. It is suggested that activation of cellular immunity would be beneficial to fully recover with the health condition of long COVID and virus-restored patients¹⁷. However, big pharma and scientists still struggle to improve neutralizing antibodies as the major function of vaccines to stop the new COVID-19 variant infection and virus recovery. Unfortunately, loss of neutralization activity and immune surveillance escape caused by virus mutations destroy the defense line of neutralizing antibodies, which raises the need for long-term T cell immunity¹⁸. According to a survey, current vaccines can provoke a high amount of neutralizing antibody but do not stop the breakthrough infection. Recently, several studies were conducted to develop broad-spectrum COVID-19 vaccines to address the issue of immune surveillance escape, including using multiple T cell epitopes, multiple spike proteins loaded on virus-like particles, conserved regions outside RBD targeting epitopes or spike S2 domains as antigens^19,20,21,22. Nearly 1 year after the COVID-19 pandemic, scientists noticed that T cell immunity played a critical protective role in COVID-19 convalescence patients and appealed to regulatory affairs that T cell immunity should be included in COVID-19 vaccine clinical trials²³. T cell vaccines are supposed to be effective against newly developed variants of COVID-19 and long COVID-19.

Eradicating virus-infected cells is the key to success in stopping breakthrough infection. For this purpose, many scientists and institutions altered the vaccine development strategy for short HLA-restricted peptides against COVID-19 virus infection and claimed broad protection from COVID-19 variants and subvariants²⁴. However, the response of CD8⁺ T cells may be lost if the HLA-I-restricted antigen contains mutations^25,26. It is suggested that HLA-restricted peptides should avoid potential mutations and glycosylation obstructions. Otherwise, it will face the same problem of activity loss for rapid evolution mutations.

Another achievable strategy for lowering breakthrough infection is to generate mucosal immunity^27,28. To strengthen COVID-19 protection, IgA antibodies were generated through mucosal immunity. It could be provoked by vaccination via nasal spray or gut delivery. It will be beneficial to generate the IgA existing in the mucosa to defend against viral infection²⁹. It has been reported that a higher level of IgA can be induced through nasal and oral administration for mucosal IgA responses³⁰. Interestingly, the only peptide without adjuvant formulation could provoke serum IgG as well as secretory IgA. It was deduced that short peptides provoke the immune response through Peyer’s patches in the intestine.

Despite the vaccination of current COVID-19 vaccines, such as mRNA and virus-based formats, the stability of vaccines is a critical issue to maintain efficacy during transportation³¹. Because of structural stability, short peptide vaccines are more stable and can be made of any formulation to fulfill the fast distribution need. To achieve a good immune response, most effective vaccines are formulated with adjuvants. Adjuvants can amplify and modulate immune responses. The CpG adjuvant is widely used in modulating the immune response to enhance Th1 cellular immunity³². However, it often causes unexpected side effects, such as autoimmune disease in a susceptible individual³³. In this study, we demonstrated the potential use of an HLA-restricted peptide COVID-19 vaccine without formulation with an adjuvant. It was shown that only short peptides are sufficient to trigger cellular and humoral immunity. It is also believed that intact immunity, including humoral and cellular immunity, will be built to protect against virus infection and that unwanted side effects can be strongly reduced.

In conclusion, we designed an HLA-I-restricted peptide based on a well-conserved S2 domain of the COVID-19 spike protein to avoid the highly mutated RBD domain and potential glycosylation-interfering with antibody binding. It might provide broad-spectrum neutralizing antibody protection against RBD-mutated COVID-19 viruses. In addition, strong and long-term memory cellular immunity, CTL, is activated by HLA-I restricted peptides to eliminate the virus-infected cells and stop producing the viruses internally. T cell vaccines provide a solution to end the COVID-19 pandemic.

HLA-restricted peptides

The HLA-restricted peptides UC-100 KLNSGDSKV and UC-152 KLNSGLSKV were designed by Vacino Biotech Taiwan from the S2 domain of the COVID-19 spike protein. The UC-100 and UC-152 peptides were synthesized from GenScript (NJ, USA) with 98% purity, and the pH value was adjusted to 7.2 ~ 7.4 for the in vitro and in vivo studies.

HLA-A201-peptide complex refolding assay

The easYmer kit (ImmunAware Aps, Copenhagen, Denmark) was used to determine the binding ability of the peptide to the HLA-A201 molecule. The detailed methods were carried out according to the kit’s manual instruction protocol. Briefly, the peptide was incubated with the β₂m-microglobulin (β₂m) light chain subunit and biotin-tagged recombinant HLA-A201 heavy chain at 18°C for 48 h to form a 500 nM folded HLA complex. The folded complexes were diluted with dilution phosphate-buffered saline (PBS) buffer containing 5% glycerol to serial concentrations of 8.9 nM, 3.0 nM and 1.0 nM. Then, 20 µL of 45-fold diluted streptavidin-coated beads (6–8 µm beads, Cat SVP-60-5, Spherotech, IL, USA) was added to each well. The beads were suspended in the solution with PE-labeled anti-human β₂m monoclonal antibody and incubated for 30 min at 4°C. The fluorescence intensity was measured by flow cytometry (BD LSRFortessa^TMX20, Franklin Lakes, NJ, USA). An HLA-A201 binding peptide NLVPMVATV (CMV pp65^495–503), which was reported to have an IC₅₀ of 45 nM for HLA-A201, was used as the positive control³⁴. Complex formation without adding peptide was used as the negative control.

In vivo study of HLA-restricted peptides

The mouse immunogenicity studies were performed by Super Laboratory Co., Ltd. (Taipei, Taiwan), and the animals were supplied by BioLASCO Taiwan Co., Ltd. (Taipei, Taiwan). For the immunogenicity study of UC-100 peptide antigen, three female BALB/c mice (7–9 weeks old) were injected intramuscularly with 3 doses of 50 µg peptide 14 days apart. For the immunogenicity study of UC-152 peptide antigen, four female BALB/c mice (7–9 weeks old) were injected intramuscularly with 3 doses of 400 µg peptide 14 days apart. For the oral ability study of UC-100 peptide antigen, five female BALB/c mice (7–9 weeks old) were orally administered 3 doses of 200 µg of peptide formulated with 1% PLCL-PEG-PLCL polymer (Sigma-Aldrich, USA). The serum was collected before the first dose of immunization (predose) and at 14 days after the last dose immunization (postdose). The serum was stored at -80°C prior to antibody measurement and functional assay. The animal protocol was reviewed and approved by the Super Laboratory company's Institutional Animal Care and Committee (IACUC #111-14c).

Measurement of IgG and IgA

To determine the total IgG and IgA levels, the IgG (Total) Mouse Uncoated ELISA Kit (Cat., No. 88-50400, Thermo Fisher Scientific, MA, USA) and Mouse IgA ELISA Kit (Cat., E-90A, Immunology Consultants Laboratory, Inc., OR, USA) were used according to the manufacturer’s protocol. For total IgG measurement, the Nunc™ MaxiSorp™ 9018 ELISA plate was coated with 100 µL of the coating buffer containing capture antibody and incubated overnight at 4°C. The plate was then washed twice with 400 µL of wash buffer. After the wells were blocked with 250 µL of blocking buffer (PBS, 1% bovine serum albumin (BSA), 0.1% Tween 20) and incubated at RT for 2 hours, the plate was washed twice. Then, 100 µL of the diluted samples and standards were added to the reaction wells. Then, 50 µL of diluted detection antibody was added to all wells. The plate was covered and incubated at RT for 2 hours. The samples were washed four times, 100 µL of substrate solution provided by the kit was added, and the samples were incubated at room temperature for 15 min. Finally, 100 µL of stop solution provided by the kit was added, and the plate was read at an optical density (OD) of 450 nm by a microplate reader (SpectraMax iD5, Molecular Devices, CA, USA). For total IgA measurement, the 4000-fold diluted samples of 100 µL were added to the IgA capture antibody-precoated plate. The procedures followed the kit’s manual instructions. The signal was read at OD 450 nm by a microplate reader (SpectraMax iD5, Molecular Devices, CA, USA).

To measure the anti-UC peptide-specific IgG, the 96-well plate was coated with 100 µL of PBS buffer containing 5 µg of UC-100 and UC-152 peptide and incubated overnight at 4°C. The following steps for anti-peptide IgG measurement were performed as described for total IgG measurement. Briefly, after incubation with anti-mouse HRP antibody, the reaction microplate was washed four times, 100 µL of TMB Solution (ab171522, Abcam, Cambridge, UK) was added, and the plate was incubated at RT for 20 min. Finally, 100 µL of Stop Solution (Cat No. 5150-0020, SeraCare, MA, USA) was added, and the plate was read at OD 450 nm by a microplate reader (SpectraMax iD5, Molecular Devices, CA, USA). For specific IgA antibody measurement, UC-100 was coated onto a 96-well microplate. The experimental procedures were performed as IgG as previously described.

To measure the anti-spike trimer IgG antibody, a mouse SARS-CoV-2 spike trimer-coated 96-well plate kit (RAS-T023, Acro biosystems, DE, USA) was used for determination. The procedure was performed according to the kit’s manual instructions. The anti-spike trimer-coated plate was added to 100 µL of diluted sample, and 100 µL dilution buffer was used as a blank. The reaction was stopped by adding 50 µL of stop solution provided by the kit and read at OD 450 nm by a microplate reader (SpectraMax iD5, Molecular Devices, CA, USA).

Th1 and Th2 cytokine release assay

Briefly, 1 x 10⁵ HLA-A201⁺ human PBMCs (STEMCELL Technologies Inc, Vancouver, British Columbia, Canada) were seeded in a 96-well microplate³⁵. The PBMCs were treated with 5 different concentrations of antigen peptides at final concentrations of 0.4, 2, 10, 50 and 250 nM and cultured with X-VIVO 15 medium for 12 days. The culture medium was refreshed every 5 days. To analyze the surface marker and intracellular cytokine levels, the suspension cells were collected and stained with anti-CD4-PerCP-Cy5.5 conjugated (Cat. 566923, BD Pharmingen, USA), IFN-γ-FITC chrome conjugated (Cat. 554700, BD Pharmingen, USA), and IL4-PE chrome conjugated (Cat. 559333, BD Pharmingen, USA) antibodies. The cytokine level in CD4⁺ T helper cells was measured by flow cytometry (BD LSRFortessa^TMX20, Franklin Lakes, NJ, U.S.)

HLA-DR-peptide engagement assay

The U-Load Dextramer kit MHC II HLA-DRB1*0101/APC (Immudex, Denmark) was used to determine the binding ability of a peptide to the HLA-DR molecule. The detailed method was performed according to the kit’s manual instruction protocol. The 10 µL MHC-II-peptide monomer was prepared by mixing the reagents of 2 µL of 1 mM diluted peptide (UC-100 and UC-152), followed by adding 3 µL of dissolved U-Load MHC-II peptide loading component and 5 µL of U-Load MHC-II (1 mg/mL). PBMCs (2 x 10⁵) were suspended in 50 µL wash buffer composed of 1% FCS in phosphate-buffered saline (PBS), pH 7.4. The 10 µL of prepared MHC dextramer as described in the instruction protocol was added to the PBMCs and incubated in the dark for 30 min at RT, followed by staining with the anti-CD4-PerCP-Cy5.5 conjugated antibody and further incubation in the dark for 20 min at RT. The cells were washed twice with 2 mL of wash buffer, and the cell pellet was resuspended in wash buffer for analysis by flow cytometry (BD LSRFortessa^TMX20, Franklin Lakes, NJ, U.S. ).

Peptide-specific CTL engagement assay

The easYmer kit (ImmunAware Aps, Denmark) was used for tetramer assay to determine the peptide-specific CTL engagement level to the peptide-HLA-A201 complex³⁶. The detailed methods were carried out according to the kit’s manual instruction protocol. Briefly, the peptide was incubated with the β₂-microglobulin (β₂m) light chain subunit and biotin-tagged recombinant HLA-A201 heavy chain for 48 h at 18°C to form a 50 µL 500 nM folded HLA complex. The tetramer was produced by incubating the equivalent of 2.1 µL of 0.2 mg/mL streptavidin-APC (Cat., 554067, BD Pharmingen, USA) and 50 µL of 500 nM folded HLA complex in the dark at 4°C overnight. PBMCs (1 x 10⁵, HLA-A201⁺) (STEMCELL Technologies Inc., Vancouver, British Columbia, Canada) were costained with the APC-labeled tetramer complex and FITC-conjugated anti-CD8 antibody (Cat., 555634, BD Pharmingen, USA) and analyzed by flow cytometry (BD LSRFortessa^TMX20, Franklin Lakes, NJ, U.S.) for the percentage of tetramer-stained CD8⁺ cells.

Cytotoxic activity assay of specific T cells

HEK293 cells expressing the SARS-CoV-2 spike (D614) (Cat No. 293-cov2-s, InvivoGen, CA, USA) were used as target cells, which were seeded into a 96-well plate³⁷. The HLA-A201⁺ PBMCs (STEMCELL Technologies Inc, Vancouver, British Columbia, Canada) were cocultured with 3,000 SARS-CoV-2 spike-expressing cells at different ratios of 27:1, 9:1, 3:1 and 1:1, and the culture was treated with UC-100 and UC-152 peptides at a final concentration of 9 nM or 27 nM for 5 days at 37°C and 5% CO₂, respectively. The cytotoxicity was measured by using the LDH-Cytox™ assay kit (Cat No. 426401, BioLegend, CA, USA). SARS-CoV-2 spike-expressing cells without PBMCs and peptide treatment were used as the negative control, while lysis buffer added to the well containing spike-expressing cells only was used as the maximum lysis control. Fifty microliters of LDH substrate was added to each well and incubated for 30 min at room temperature. Finally, the absorbance of OD at 490 nm was read by a microplate reader (EnVision Xcite Multilable Reader, PerkinElmer, MA, USA). The percentage of specific cell lysis was calculated according to the manufacturer’s instructions.

Complement-dependent cytotoxicity assay

To determine the function of the antibody induced by the UC peptide, the complement cytotoxicity was measured on SARS-CoV-2 spike (D614)-expressing HEK293 cells (Cat No. 293-cov2-s, InvivoGen CA, USA)³⁸. The cytotoxicity mediated by the antibody was measured by using an LDH-Cytox™ assay kit (BioLegend, CA, USA). The 4 x 10³ SARS-CoV-2 spike-expressing cells were suspended in 45 µL of serum-free DMEM in each well of a 96-well microplate. The serum samples were serially diluted for 9 dilutions with serum-free DMEM starting at a ratio of 1:20 with 2-fold to 5120. Five microliters of diluted serum was added to the wells containing SARS-CoV-2-expressing cells and incubated for 15 min at 37°C and 5% CO₂. A final concentration of 10% of the baby rabbit complement (CL3441-S50-R, Cedarlane, Ontario, Canada) was added to each well and incubated for an additional 30 min at 37°C and 5% CO₂. The plate was then cooled at room temperature for 15 min for measurement of the cytotoxicity by LDH release. The sample wells containing serum were used as background controls, while lysis buffer was added to the sample wells containing SARS-CoV-2-expressing cells, and complement was used as the maximum lysis control. Then, 50 µL of LDH substrate was added to each well and incubated for 30 min at room temperature. Finally, the absorbance of OD at 490 nm was read by a microplate reader (EnVision Xcite Multilable Reader, PerkinElmer, MA, USA). The percentage of specific cell lysis was calculated according to the manufacturer’s instructions.

Statistical analysis

The statistics were compiled by the unpaired one-tailed Student’s t-test embedded in Prism 5 (GraphPad Software Inc., San Diego, CA, USA) in the measurement of IgG antibody, IgA antibody, tetramer and dextramer. A p value < 0.05 was considered significant (*p < 0.05, **p < 0.01, and ***p < 0.001).

Data availability

The predicted binding affinity of peptides to MHC-I was analyzed with SMM on the Immune Epitope Database website (https://www.iedb.org/). Source data are provided with this paper.

Acknowledgements

We thank Super Laboratory Co., Ltd. (Taiwan) for animal operation and serum collection in this study.

Author contributions

J.M.C. and Y.R.L. designed the HLA-restricted peptide and supervised this study. C. W.L. performed the mechanistic immune response of the in vitro assays and complement activity of the induced ex vivo assay. I.H.L. performed the measurement of the level of total antibody, specific antibody against peptide and spike trimer protein. All authors participated in analyzing the data, and J.M.C. and Y.R.L. wrote the manuscript, and all authors helped revise the manuscript.

Competing interests

This research was financially supported by Vacino Biotech Co., Ltd. (Taiwan). Vacino Biotech has patents (US 17/726,688, PCT/CN2022/089266, ROC 111115497) concerning the therapeutic compounds used in this study, where Yi-Ru Lee and Jia-Ming Chang are also the authors of the patents. All authors are employees of Vacino Biotech company.

Kyriakidis, N.C., López-Cortés, A., González, E.V., Grimaldos, A. B. & Prado, E. O. SARS-CoV-2 vaccines strategies: A comprehensive review of phase 3 candidates. npj Vaccines6, 28 (2021).
Sansone, M., Studahl, M., Berg, S., Gisslén, M. & Sundell, N. Severe multisystem inflammatory syndrome (MIS-C/A) after confirmed SARS-CoV-2 infection: A report of four adult cases. Infect. Dis.54, 378-383 (2022).
Stafie, C. S. et al. Pathogenic Connections in Post-COVID Conditions: What Do We Know in the Large Unknown? A Narrative Review. Viruses. 14, 1686 (2022).
Hachmann, N. P. et al. Neutralization escape by SARS-CoV-2 Omicron subvariants BA.2.12.1, BA.4, and BA.5. N. Engl. J. Med. 387, 86-88 (2022).
Hu, J. et al. Reduced neutralization of SARS-CoV-2 B.1.617 variant by convalescent and vaccinated sera. Genes Dis. 9, 1290-1300 (2022).
Morens, D. M., Taubenberger, J. K. & Fauci, A. S. Universal coronavirus vaccines-An urgent need. N. Engl. J. Med.386, 297-299 (2022).
Wherry E. J. & Barouch D. H. T cell immunity to COVID-19 vaccines. Science377, 821-822 (2022).
Kumar S., Karuppanan K. & Subramaniam G. Omicron (BA.1) and sub-variants (BA.1.1, BA.2, and BA.3) of SARS-CoV-2 spike infectivity and pathogenicity: A comparative sequence and structural-based computational assessment. J. Med. Virol. 94, 4780-4791 (2022).
Vashishtha V. et al. Looking to the future: is a universal coronavirus vaccine feasible? Expert Rev. Vaccines21, 277-280 (2022).
Le Bert, N. et al. SARS-CoV-2-specific T cell immunity in cases of COVID-19 and SARS, and uninfected controls. Nature584, 457-464 (2020).
Ng, O.-W. et al. Memory T cell responses targeting the SARS coronavirus persist up to 11 years post-infection. Vaccine34, 2008-2014 (2016).
Moss, P. The T cell immune response against SARS-CoV-2. Nat. Immunol.23, 186–193 (2022).
Dolgin, E. Nat. Biotechnol.40, 3-4 (2022).
Planas, D. et al. Considerable escape of SARS-CoV-2 Omicron to antibody neutralization. Nature602, 671–675 (2022).
Lipsitch, M., Krammer, F., Regev-Yochay, G., Lustig, Y. & Balicer, R. D. SARS-CoV-2 breakthrough infections in vaccinated individuals: measurement, causes and impact. Nat. Rev. Immunol.22, 57–65 (2022).
Callaway E. Naturehttps://www.nature.com/articles/d41586-022-02121-z (2022).
Wiech, M. et al. Remodeling of T Cell dynamics during long COVID is dependent on severity of SARS-CoV-2 infection. Front. Immunol. 13, 886431 (2022).
Riou, C et al. Escape for recognition of SARS-CoV-2 variant spike epitopes but overall preservation of T cell immunity. Sci. Transl. Med.14, eabj6824 (2022).
Rouzbahani, A.K., Kheirandish, F. & Hosseini, S.Z. Design of a multi-epitope-based peptide vaccine against the S and N proteins of SARS-COV-2 using immunoinformatics approach. Egypt. J. Med. Hum. Genet.23, 16 (2022).
Dolgin, E. Nat. Rev. Drug Discov. 21, 324-326 (2022)
Fang Y. et al. An antibody that neutralizes SARS-CoV-1 and SARS-CoV-2 by binding to a conserved spike epitope outside the receptor binding motif. Sci. Immunol. https://www.science.org/doi/10.1126/sciimmunol.abp9962 (2022)
Ng, K. W. et al. SARS-CoV-2 S2-targeted vaccination elicits broadly neutralizing antibodies. Sci. Transl. Med.14, eabn3715 (2022).
Wherry, E. J. et al. Letter to FDA- T-cell assessment in vaccine studies_4-21-22_FINAL.pdf. 2022. Available: https://drive.google.com/file/d/1OPfStqOnuKAEUkrjfFUouXMjDB_-tnmV/edit
Tarke, A. et al. Comprehensive analysis of T cell immunodominance and immunoprevalence of SARS-CoV-2 epitopes in COVID-19 cases. Cell Rep. Med.2, 100204 (2021).
Agerer, B. et al. SARS-CoV-2 mutations in MHC-I-restricted epitopes evade CD8 ⁺ T cell responses. Sci. Immunol.6, eabg6461 (2021).
Hamelin, D. J. et al., The mutational landscape of SARS-CoV-2 variants diversifies T cell targets in an HLA-supertype dependent manner. Cell Syst.13, 143–157 (2022).
Lavelle, E. C. & Ward, R. W. Mucosal vaccines - fortifying the frontiers. Nat. Rev. Immunol.22, 236-250 (2022).
Russell, M. W., Moldoveanu, Z., Ogra, P. L. & Mestecky J. Mucosal immunity in COVID-19: a neglected but critical aspect of SARS-CoV-2 infection. Front. Immunol.11, 611337 (2020).
Sheikh-Mohamed, S. et al. Systemic and mucosal IgA responses are variably induced in response to SARS-CoV-2 mRNA vaccination and are associated with protection against subsequent infection. Mucosal Immunol.15, 799–808 (2022).
Boyaka, P. N. Inducing mucosal IgA: A challenge for vaccine adjuvants and delivery systems. J. Immunol.199, 9–16 (2017)
Uddin, M. N. & Roni, M. A. Challenges of storage and stability of mRNA-based COVID-19 vaccines. Vaccines (Basel)9, 1033 (2021).
Millan C. L. B., Weeratna, R., Krieg, A. M., Siegrist, C.-A. & Davis H. L. CpG DNA can induce strong Th1 humoral and cell-mediated immune responses against hepatitis B surface antigen in young mice. Proc. Natl. Acad. Sci. U.S.A.95, 15553–15558 (1998).
Segal, B. M. Chang, J. T. & Shevach, E. M. CpG oligonucleotides are potent adjuvants for the activation of autoreactive encephalitogenic T cells in vivo. J. Immunol.164, 5683-5688. (2000).
Hassan C. et al. Naturally processed non-canonical HLA-A*02:01 presented peptides. J. Biol. Chem. 290, 2593-2603 (2015)
Tapia-Calle, G. et al. A PBMC-based system to assess human T cell response to influenza vaccine candidates in vitro. Vaccines (Basel) 7, 181 (2019).
Bonté, P.-E. et al. Single-cell RNA-seq-based proteogenomics identifies glioblastoma-specific transposable elements encoding HLA-I-presented peptides. Cell Rep.39, 110916 (2022).
Babaer, D., Zheng, M., Ivy M. T., Zent R. & Tiriveedhi, V. Methylselenol producing selenocompounds enhance the efficiency of mammaglobin‑A peptide vaccination against breast cancer cells. Oncol. Lett.18, 6891-6898 (2019).
Osada, T. et al. Polyfunctional anti-human epidermal growth factor receptor 3 (anti-HER3) antibodies induced by HER3 vaccines have multiple mechanisms of antitumor activity against therapy resistant and triple negative breast cancers. Breast Cancer Res.20, 90 (2018).

Yes there is potential Competing Interest. This research was financially supported by Vacino Biotech, Co. Ltd (Taiwan). Vacino Biotech have patents (US 17/726,688, PCT/CN2022/089266, ROC 111115497) concerning the therapeutic compounds used in this study, where Yi-Ru Lee and Jia-Ming Chang are also the authors of the patents. All authors are employee of Vacino Biotech company.

Sourcedatafinal.xlsx
Data availability-01
nreditorialpolicychecklist.pdf

Download PDF

Version 1

posted

You are reading this latest preprint version

T cell immunity of the nonadjuvanted HLA-restricted peptide COVID-19 vaccine

Status:

Version 1

Abstract

Figures

Introduction

Results

The UC peptides could be primed onto the CTLs and engaged with the corresponding CTLs

UC peptides polarized the Th1 cytokine release of human PBMCs

UC peptides could directly activate the specific lysis activity of CTLs

The UC peptide-induced antibody could activate complement to lyse epitope-bearing cells

The oral administration of UC peptides provokes immunogenicity in mice

Discussion

Methods

HLA-restricted peptides

HLA-A201-peptide complex refolding assay

In vivo study of HLA-restricted peptides

Measurement of IgG and IgA

Th1 and Th2 cytokine release assay

HLA-DR-peptide engagement assay

Peptide-specific CTL engagement assay

Cytotoxic activity assay of specific T cells

Complement-dependent cytotoxicity assay

Statistical analysis

Declarations

References

Additional Declarations

Supplementary Files

Status:

Version 1