Cross-protection induced by highly conserved human B, CD4+, and CD8+ T-cell epitopes-based vaccine against severe infection, disease, and death caused by multiple SARS-CoV-2 variants of concern

Background The coronavirus disease 2019 (COVID-19) pandemic has created one of the largest global health crises in almost a century. Although the current rate of Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections has decreased significantly, the long-term outlook of COVID-19 remains a serious cause of morbidity and mortality worldwide, with the mortality rate still substantially surpassing even that recorded for influenza viruses. The continued emergence of SARS-CoV-2 variants of concern (VOCs), including multiple heavily mutated Omicron sub-variants, has prolonged the COVID-19 pandemic and underscores the urgent need for a next-generation vaccine that will protect from multiple SARS-CoV-2 VOCs. Methods We designed a multi-epitope-based coronavirus vaccine that incorporated B, CD4+, and CD8+ T- cell epitopes conserved among all known SARS-CoV-2 VOCs and selectively recognized by CD8+ and CD4+ T-cells from asymptomatic COVID-19 patients irrespective of VOC infection. The safety, immunogenicity, and cross-protective immunity of this pan-variant SARS-CoV-2 vaccine were studied against six VOCs using an innovative triple transgenic h-ACE-2-HLA-A2/DR mouse model. Results The pan-variant SARS-CoV-2 vaccine (i) is safe , (ii) induces high frequencies of lung-resident functional CD8+ and CD4+ TEM and TRM cells , and (iii) provides robust protection against morbidity and virus replication. COVID-19-related lung pathology and death were caused by six SARS-CoV-2 VOCs: Alpha (B.1.1.7), Beta (B.1.351), Gamma or P1 (B.1.1.28.1), Delta (lineage B.1.617.2), and Omicron (B.1.1.529). Conclusion A multi-epitope pan-variant SARS-CoV-2 vaccine bearing conserved human B- and T- cell epitopes from structural and non-structural SARS-CoV-2 antigens induced cross-protective immunity that facilitated virus clearance, and reduced morbidity, COVID-19-related lung pathology, and death caused by multiple SARS-CoV-2 VOCs.


Introduction
While the Wuhan Hu1 variant of SARS-CoV-2 is the ancestral reference virus, Alpha (lineage B.1.1.7),Beta (lineage B.1.351),Gamma (lineage B. 1.1.28),and Delta (lineage B.1.617.2) variants of concern (VOCs) subsequently emerged in the United Kingdom, South Africa, Brazil, and India, respectively, between 2020 and 2022 (1).The most recent SARS CoV-2 variants, including multiple heavily mutated Omicron (B.1.1.529)sub-variants, have prolonged the COVID-19 pandemic (2)(3)(4)(5)(6).These new variants emerged beginning December 2020 at a much higher rate, with the accumulation of two mutations per month, and exerting strong selective pressure on the immunologically important SARS-CoV-2 genes (7).The Alpha, Beta, Gamma, Delta, and Omicron variants are defined as VOCs based on their high transmissibility associated with increased hospitalizations and deaths (8).This is a result of reduced neutralization by antibodies generated by previous variants and/or by the first-generation COVID-19 vaccines, together with failures of treatments and diagnostics (9,10).Dr. Peter Marks, Director/CBER (Center for Biologics Evaluation and Research) for the FDA recently outlined the need for a superior next-generation vaccine that will protect from multiple SARS-CoV-2 VOCs (11, 12).
Besides SARS CoV-2 variants, two additional coronaviruses from the severe acute respiratory syndrome (SARS)-like betacoronavirus (sarbecovirus) lineage, SARS coronavirus (SARS-CoV-1) and MERS-CoV, have caused epidemics and pandemics in humans over the past 20 years (13).In addition, the discovery of diverse sarbecoviruses in bats together with the frequent "jumping" of these zoonotic viruses from bats to intermediate animal hosts raises the possibility of another COVID-19-like pandemic in the future (14-19).Hence, there is an urgency to develop a pre-emptive universal pan-variant SARS-CoV-2 vaccine to protect against all SARS-CoV-2 variants, SARS-CoV, MERS-CoV, and other zoonotic Sarbecoviruses with the potential to jump from animals into humans.

Triple transgenic mice immunization with SARS-CoV-2 conserved peptides and infection
The

Human study population cohort and HLA genotyping
In this study, we have included 210 subjects from a pool of over 682 subjects.Written informed consent was obtained from participants before inclusion.The subjects were categorized as mild to severe COVID-19 groups and have undergone treatment at the University of California Irvine Medical Center between July 2020 and July 2022 (Institutional Review Board protocol no.2020-5779).SARS-CoV-2 positivity was defined by a positive RT-PCR on nasopharyngeal swab samples.All the subjects were genotyped by PCR for class I HLA-A*02:01 and class II HLA-DRB1*01:01 among the 682 patients (and after excluding a few for which the given amount of blood was insufficient -i.e., <6ml), we ended up with 210 that were genotyped for HLA-A*02:01 + or/and HLA-DRB1*01:01 + (35,36).Based on the severity of symptoms and ICU admission/intubation status, the subjects were divided into five broad severity categories, namely, Severity 5, patients who died from COVID-19 complications; Severity 4, infected COVID-19 patients with severe disease that were admitted to the intensive care unit (ICU) and required ventilation support; Severity 3, infected COVID-19 patients with severe disease that required enrollment in ICU, but without ventilation support; Severity 2, infected COVID-19 patients with moderate symptoms that involved a regular hospital admission; Severity 1, infected COVID-19 patients with mild symptoms; and Severity 0, infected individuals with no symptoms.Demographically, the 210 patients included were from mixed ethnicities [Hispanic (34%), Hispanic Latino (29%), Asian (19%), Caucasian (14%), Afro-American (3%), and Native Hawaiian and Other Pacific Islander descent (1%)].

Sequence comparison among variants of SARS-CoV-2 and animal CoV strains
We retrieved nearly 8.5 million human SARS-CoV-2 genome sequences from the GISAID database representing countries from North America, South America, Central America, Europe, Asia, Oceania, Australia, and Africa.All the sequences included in this study were retrieved either from the NCBI GenBank (www.ncbi.nlm.nih.gov/nuccore) or GISAID (www.gisaid.org).Multiple sequence alignment was performed keeping SARS-CoV-2-Wuhan-Hu-1 (MN908947.3)protein sequence as a reference against all the SARS-CoV-2 VOCs, common cold, and animal CoV strains.The sequences were aligned using the high throughput alignment tool DIAMOND (37).This comprised of all the VOCs and VBMs of SARS

SARS-CoV-2 CD8 + and CD4 + T-cell epitope prediction
Epitope prediction was performed considering the spike glycoprotein (YP_009724390.1) for the reference SARS-CoV-2 isolate, Omicron BA.2.The reference spike protein sequence was used to screen CD8 + T cell and CD4 + T-cell epitopes.The tools used for CD8 + T-cell-based epitope prediction were SYFPEITHI, MHC-I binding predictions, and Class I Immunogenicity.Of these, the latter two were hosted on the IEDB platform.We used multiple databases and algorithms for the prediction of CD4 + T-cell epitopes, namely, SYFPEITHI, MHC-II Binding Predictions, Tepitool, and TEPITOPEpan.For CD8 + T cell epitope prediction, we selected the 5 most frequent HLA-A class I alleles (HLA-A*01:01, HLA-A*02:01, HLA-A*03:01, HLA-A*11:01, HLA-A*23:01) with nearly 80% coverage of the world population, regardless of race and ethnicity, using a phenotypic frequency cutoff ≥ 6%.Similarly, for CD4 + T-cell epitope prediction, HLA-DRB1*01:01, HLA-DRB1*11:01, HLA-DRB1*15:01, HLA-DRB1*03:01, and HLA-DRB1*04:01 alleles with population coverage of 60% were selected.Subsequently, using NetMHC, we analyzed the SARS-CoV-2 protein sequence against all the MHC-I and MHC-II alleles.Epitopes with 9-mer lengths for MHC-I and 15-mer lengths for MHC-II were predicted.Subsequently, the peptides were analyzed for binding stability to the respective HLA allotype.Our stringent epitope selection criteria were based on picking the top 1% epitopes focused on prediction percentile scores.N and O glycosylation sites were screened using NetNGlyc 1.0 and NetOGlyc 4.0 prediction servers, respectively.

Population-coverage-based T-cell epitope selection
For a robust epitope screening, we evaluated the conservancy of CD8 + T cell, CD4 + T cell, and B-cell epitopes within spike glycoprotein of Human-SARS-CoV-2 genome sequences representing North America, South America, Africa, Europe, Asia, and Australia.As of 20 April 2022, the GISAID database extrapolated 8,559,210 human-SARS-CoV-2 genome sequences representing six continents.Population coverage calculation (PPC) was carried out using the Population Coverage software hosted on the IEDB platform.PPC was performed to evaluate the distribution of screened CD8  ) that we formerly identified were selected as described previously (33).The Epitope Conservancy Analysis tool was used to compute the degree of identity of CD8 + T-cell and CD4 + T-cell epitopes within a given protein sequence of SARS-CoV-2 set at 100% identity level (33).Peptides were synthesized as previously described (21st Century Biochemicals, Inc, Marlborough, MA).The purity of peptides determined by both reversed-phase highperformance liquid chromatography and mass spectroscopy was over 95%.Peptides were first diluted in DMSO and later in PBS (1 mg/mL concentration).The helper T-lymphocyte (HTL) epitopes for the selected SARS-CoV-2 proteins were predicted using the MHC-II epitope prediction tool from the Immune Epitope Database (IEDB, http://tools.iedb.org/mhcii/).Selected epitopes had the lowest percentile rank and IC 50 values.Additionally, the selected epitopes were checked by the IFN epitope server (http:// crdd.osdd.net/raghava/ifnepitope/)for the capability to induce Th1 type immune response accompanied by IFN-ϒ production.Cytotoxic T-lymphocyte (CTL) epitopes for the screened proteins were predicted using the NetCTL1.2server (http://www.cbs.dtu.dk/services/NetCTL/).

SARS-CoV-2 B-cell epitope prediction
Linear B-cell epitope predictions were carried out on the spike glycoprotein (S), the primary target of B-cell immune responses for SARS-CoV.We used the BepiPred 2.0 algorithm embedded in the B-cell prediction analysis tool hosted on the IEDB platform.For each protein, the epitope probability score for each amino acid and the probability of exposure was retrieved.Potential B-cell epitopes were predicted using a cutoff of 0.55 (corresponding to a specificity >0.81 and sensitivity <0.3) and considering sequences having more than five amino acid residues.This screening process resulted in eight B-cell peptides.These epitopes represent all the major nonsynonymous mutations reported among the SARS-CoV-2 variants.One B-cell epitope (S 439-482 ) was observed to possess the maximum number of variant-specific mutations.Structure-based antibody prediction was performed using Discotope 2.0, and a positivity cutoff greater than −2.5 was applied (corresponding to specificity ≥ 0.80 and sensitivity <0.39), using the SARS-CoV-2 spike glycoprotein structure (PDB ID: 6M1D).

TaqMan quantitative polymerase reaction assay for the screening of SARS-CoV-2 variants in COVID-19 patients
We utilized a laboratory-developed modification of the CDC SARS-CoV-2 RT-PCR assay, which received Emergency Use Authorization by the FDA on April 17th 2020.(https:// www.fda.gov/media/137424/download[accessed 24 March 2021]).38).The probe overlaps with the sequences that contain amino acids 69 -70; therefore, a negative result for this assay predicts the presence of deletion S-D69-70 in the sample.Using a similar strategy, a primer/ probe set that targets the deletion S-D242-244 was designed and was run in the same reaction with S-D69-70.In addition, three separate assays were designed to detect spike mutations S-501Y, S-484K, and S-452R, and wild-type positions S-501N, S-484E, and S-452L.

Mutation screening assays
Briefy, 5 µl of the total nucleic acid eluate was added to a 20-µl total volume reaction mixture (1× TaqPath 1-Step RT-qPCR Master Mix, CG [Thermo Fisher Scientific, Waltham, MA], with 0.9 mM each primer and 0.2 mM each probe).The RT-PCR was carried out using the ABI StepOnePlus thermocycler (Life Technologies, Grand Island, NY).The S-N501Y, S-E484K, and S-L452R assays were carried out under the following running conditions: 25°C for 2 min, then 50°C for 15 min, followed by 10 min at 95°C and 45 cycles of 95°C for 15 s and 65°C for 1 min.The D 69-70/D242-244 assays were run under the following conditions: 25°C for 2 min, then 50°C for 15 min, followed by 10 min at 95°C and 45 cycles of 95°C for 15 s and 60°C for 1 min.Samples displaying typical amplification curves above the threshold were considered positive.Samples that yielded a negative result or results in the S-D69-70/D242-244 assays or were positive for S-501Y P2, S-484K P2, and S-452R P2 were considered screen positive and assigned to a VOC.

Neutralizing antibody assays for SARS-CoV-2
Serially diluted heat-inactivated plasma (1:3) and 300 pfu of SARS-CoV-2 variants are combined in Dulbecco's modified Eagle's medium (DMEM) and incubated at 37°C 5% CO 2 for 30 min.After neutralization, the antibody-virus inoculum was transferred onto Vero E6 cells (ATCC C1008) and incubated at 34°C 5% CO 2 for 1 h.The cells were then fixed with 10% neutral buffered formalin and incubated at −20°C for 10 min followed by 20 min at room temperature.Plates were developed with True Blue HRP substrate and imaged on an ELISpot reader.The half maximum inhibitory concentration (IC50) was calculated using normalized counted foci.

Histology of animal lungs
Mouse lungs were preserved in 10% neutral buffered formalin for 48 h before transferring to 70% ethanol.The tissue sections were then embedded in paraffin blocks and sectioned at 8-mm thickness.Slides were deparaffinized and rehydrated before staining for hematoxylin and eosin for routine immunopathology.IHC was performed on mice lung tissues probed with SARS/SARS-CoV-2 Coronavirus NP Monoclonal Antibody (B46F) (Product No. MA1-7404) at a dilution of 1:100.The antibody showed significant staining in lung tissues of non-immunized, SARS-CoV-2-infected mice when compared to the tissues of the vaccinated group of mice.This method was meant to demonstrate the relative expression of the Nucleocapsid protein between non-immunized Mock and immunized samples.Further CD8 + T-cell and CD4 + T-cellspecific staining were performed to identify the T-cell infiltration among the immunized and Mock groups.

Peripheral blood mononuclear cells isolation and T cell stimulation
Peripheral blood mononuclear cells (PBMCs) from COVID-19 patients were isolated from the blood using Ficoll (GE Healthcare) density gradient media and transferred into 96-well plates at a concentration of 2.5 × 10 6 viable cells per ml in 200 µl (0.5 × 10 6 cells per well) of RPMI-1640 media (Hyclone) supplemented with 10% (v/v) FBS (HyClone), sodium pyruvate (Lonza), L-glutamine, non-essential amino acids, and antibiotics (Corning).A fraction of the blood was kept separated to perform HLA genotyping of the patients and select only the HLA-A*02:01-and/or DRB1*01: 01positive individuals.Subsequently, cells were then stimulated with 10 µg/ml of each one of the 22 individual T-cell peptide epitopes (16 CD8 + T-cell peptides and 6 CD4 + T-cell peptides) and incubated in humidified 5% CO 2 at 37°C.Post-incubation, cells were stained by flow cytometry analysis or transferred in IFN-g ELISpot plates.The same isolation protocol was followed for healthy donor (HD) samples obtained in 2018.PBMC samples were kept frozen in liquid nitrogen in 10% FBS in DMSO.Upon thawing, HD PBMCs were stimulated in the same manner for the IFN-g ELISpot technique.Furthermore, to evaluate the immunogenicity of conserved SARS-CoV-2 CD8 + and CD4 + T-cell epitopes in triple transgenic HLA-A*02:01/HLA-DRB1*01:01-hACE-2 mice, mononuclear cells from lung tissues were collected 14 days post-infection.ELISpot assay was performed as described previously (33,39).

Flow cytometry analysis
After 72 h of stimulation with each SARS-CoV-2 class I or class II restricted peptide, PBMCs (0.5 × 10 6 cells) from 147 patients were stained for the detection of surface markers and subsequently analyzed by flow cytometry.First, the cells were stained with a live/dead fixable dye (Zombie Red dye, 1/800 dilution -BioLegend, San Diego, CA) for 20 min at room temperature, to exclude dying/ apoptotic cells.Cells were stained for 45  Subsequently, we used anti-human antibodies for surface marker staining: anti-CD62L, anti-CD69, anti-CD4, anti-CD8, and anti-IFN-g.mAbs against these various cell markers were added to the cells in phosphate-buffered saline (PBS) containing 1% FBS and 0.1% sodium azide (fluorescence-activated cell sorter [FACS] buffer) and left for 30 min at 4°C.At the end of the incubation period, the cells were washed twice with FACS buffer and fixed with 4% paraformaldehyde (PFA, Affymetrix, Santa Clara, CA).A total of ∼200,000 lymphocyte-gated PBMCs (140,000 alive CD45 + ) were acquired by Fortessa X20 (Becton Dickinson, Mountain View, CA) and analyzed using FlowJo software (TreeStar, Ashland, OR).

Enzyme-linked immunosorbent assay
Serum antibodies specific for epitope peptides and SARS-CoV-2 proteins were detected by ELISA.The 96-well plates (Dynex Technologies, Chantilly, VA) were coated with 0.5 mg peptides and 100 ng S or N protein per well at 4°C overnight, respectively, and then washed three times with PBS and blocked with 3% BSA (in 0.1% PBST) for 2 h at 37°C.After blocking, the plates were incubated with serial dilutions of the sera (100 ml/well, in twofold dilution) for 2 h at 37°C.The bound serum antibodies were detected with HRP-conjugated goat anti-mouse IgG and chromogenic substrate TMB (Thermo Fisher, Waltham, MA).The cut-off for seropositivity was set as the mean value plus three standard deviations (3SD) in HBc-S control sera.The binding of the epitopes to the sera of SARS-CoV-2-infected samples was detected by ELISA using the same procedure; 96-well plates were coated with 0.5 mg peptides, and sera were diluted at 1:50.All ELISA studies were performed at least twice.

Data and code availability
The human-specific SARS-CoV-2 complete genome sequences were retrieved from the GISAID database, whereas the SARS-CoV-2 sequences for pangolin (Manis javanica) and bat (Rhinolophus affinis, Rhinolophus malayanus) were retrieved from NCBI.Genome sequences of previous strains of SARS-CoV for humans, bats, civet cats, and camels were retrieved from the NCBI GenBank.
Next, we determined whether the highly conserved "universal" CD8  Similarly, higher frequencies of functional IFN-g-producing CD4 + T cells ASYMP COVID-19 patients (mean SFCs > 25 per 1 × 10 6 pulmonary immune cells) were detected, irrespective of infection with Beta (p < 0.5, Figure 1C, left panel) or Omicron (p < 0., Figure 1C, right panel) variants, whereas reduced frequencies of IFN-g-producing CD4 + T cells were detected in SYMP COVID-19 patients, irrespective of infection with Beta (p < 0.5, Figure 1C, left panel) or Omicron (p < 0., Figure 1C, right panel) variants.This observation was consistent regardless of whether the CD4 + T-cell targeted epitopes were from structural or non-structural SARS-CoV-2 protein antigens.Our results suggest that strong CD4 + T-cell responses specific to selected "universal" SARS-CoV-2 epitopes were commonly associated with better COVID-19 outcomes.In contrast, low SARS-CoV-2-specific CD4 + T-cell responses were more commonly associated with severe disease onset.
Taken together, these results (1) demonstrate an important role of SARS-CoV-2-specific CD4 + and CD8 + T cells directed against highly conserved structural and non-structural SARS-CoV-2 epitopes in protection from severe COVID-19 symptoms, (2) highlight the potential importance of these highly conserved "asymptomatic" epitopes in mounting protected CD4 + and CD8 + T cell responses against multiple SARS-CoV-2 VOCs, and (3) support targeting these conserved epitopes with a vaccine.
A pan-variant SARS-CoV-2 vaccine composed of a mixture of conserved "asymptomatic" CD4 + and CD8 + T cell epitopes provides robust protection against infection and disease caused by six SARS-CoV-2 variants of concern We next used a prototype pan-variant SARS-CoV-2 vaccine composed of a mixture of 6 conserved "asymptomatic" CD4 + T-cell epitopes and 16 conserved "asymptomatic" CD4 + and CD8 + T-cell epitopes that span the whole SARS-CoV-2 genome (33).We focused mainly on CD4 + and CD8 + T-cell epitopes that show immunodominance selectively in SYMP COVID-19 patients infected with various SARS-CoV-2 VOCs.
Throat swabs were collected from the vaccinated and mockvaccinated groups of mice on days 2, 4, 6, 8, 10, and 14 post-infection (p.i.) and were processed to detect the viral RNA copy number by qRT-PCR (Figure 2D).Compared to the viral RNA copy number detected from the mock-vaccinated group of mice, we detected a statistically significant decrease in the viral RNA copy number among vaccinated groups of mice on day 4 p. for SARS-CoV-2 WA/USA2020 (p = 0.02) (Figure 2D).These results demonstrate that the pan-variant SARS-CoV-2 vaccine conferred significant protection from virus replication against SARS-CoV-2 variants and supports the hypothesis that a broad anti-viral effect following immunization with asymptomatic B and CD4 + and CD8 + T-cell epitopes carefully selected as being highly conserved from multiple SARS-CoV-2 variants.

Immunization with the pan-variant SARS-CoV-2 vaccine bearing conserved epitopes reduced COVID-19-related lung pathology and virus replication associated with increased infiltration of CD8 + and CD4 + T cells in the lungs
Hematoxylin and eosin staining of lung sections at day 14 p.i. showed a significant reduction in COVID-19-related lung pathology in the mice immunized with conserved pan-variant SARS-CoV-2 vaccine compared to mock-vaccinated mice (Figure 3A).This reduction in lung pathology was observed for all six SARS-CoV-2 variants: USA-WA1/2020, Alpha (B.1.1.7),Beta (B.1.351),Gamma (P.1), Delta (B.1.617.2), and Omicron (B.1.1.529)(Figure 3A).We further performed SARS-CoV-2 Nucleocapsid Antibody-Based Immunohistochemistry (IHC) staining on lung tissues obtained from vaccinated and mock-vaccinated groups of mice infected with SARS-CoV-2 variants.We detected significantly lower antibody staining in the lung tissues of the vaccinated compared mock-vaccinated group of mice following infection with each of the six SARS-CoV-2 variants of concern.This indicated higher expression of the target viral proteins in the lungs of the mock-vaccinated compared to the vaccinated group of mice (Figure 3B).Furthermore, IHC staining was performed to compare the infiltration CD8 + and CD4 + T cells into lung tissues of vaccinated and mock-vaccinated mice infected with various SARS-CoV-2 variants.We observed a significant increase in the infiltration of both CD8 + T cells (Figure 3C) and CD4 + T cells (Figure 3D) in the lungs of vaccinated mice compared to mockvaccinated mice 14 days following infection with each of the six variants.
Taken together, these results indicate that immunization with the pan-variant SARS-CoV-2 vaccine bearing conserved epitopes induced cross-protective CD8 + and CD4 + T cells that infiltrated the lungs, faciltated clearance of virus, and reduced COVID-19-related lung pathology following infection with various multiple SARS-CoV-2 variants.

Increased frequencies of lung-resident functional CD8 + and CD4 + T EM and T RM cells induced by the pan-variant SARS-CoV-2 vaccine are associated with protection against multiple SARS-CoV-2 variants
To determine whether increased frequencies of lung-resident functional CD8 + and CD4 + T cells induced by the pan-variant SARS-CoV-2 vaccine are associated with protection against multiple SARS-CoV-2 variants, we used flow cytometry to compared the frequencies of IFN-g CD8 + T cells and CD69 CD8 + T cells (Figure 4A), IFN-g CD4 + T cells and CD69 CD4 + T cells (Figure 4B) in cell suspensions from the lungs of vaccinated versus mock-vaccinated groups of mice.
were determined in the lung tissues of vaccinated and mockvaccinated mice after challenge with each of six different SARS-CoV-2 variants of concern.
Taken together, these results (1) confirm that immunization with the pan-variant SARS-CoV-2 vaccine bearing conserved epitopes induced high frequencies of functional CD8 + T cells that infiltrated the lungs and were associated with cross-protection against multiple SARS-CoV-2 variants; (2) demonstrate that increased SARS-CoV-2 epitope-specific IFN-g-producing CD8 + T cells in the lungs of vaccinated triple transgenic HLA-A*02:01/ HLA-DRB1*01:01-hACE-2 mice were associated with protection from multiple variants of concern.In contrast, low frequencies of lung-resident SARS-CoV-2-specific IFN-g-producing CD8 + T cells were associated with severe disease onset in mock-vaccinated triple transgenic HLA-A*02:01/HLA-DRB1*01:01-hACE-2 mice.In this report, we suggest an important role for functional lung-resident SARS-CoV-2-specific CD8 + T cells specific to highly conserved "universal" epitopes from structural and non-structural antigens in cross-protection against SARS-CoV-2 VOCs.
Increased SARS-CoV-2 epitopes-specific IFN-g-producing CD4 + T cells in the lungs of vaccinated mice in comparison to mock-vaccinated mice  producing CD4 + T cells using ELISpot, to determine whether the functional lung-resident CD4 + T cells are specific to SARS-CoV-2 (Figure 6).Overall, we detected a significant increase in the number of IFNg-producing CD4 + T cells in the lungs of protected mice that received the pan-variant SARS-CoV-2 vaccine compared to non-protected mock-vaccinated mice (mean SFCs > 25 per 0.5 × 10 6 pulmonary immune cells), irrespective of the SARS-CoV-2 VOCs: WA/USA2020 (Figure 6A .We observed 100% conservancy in three of our earlier predicted B-cell epitopes, namely, S 287-317 , S 524-558 , and S 565-598 (Supplementary Figure S3).The antibody titer specific to each of the nine "universal" B-cell epitopes was determined by ELISA in COVID-19 patients infected with multiple SARS-CoV-2 variants of concern (Supplementary Figure S4, left panel) and in vaccinated and mock-vaccinated triple transgenic HLA-A*02:01/HLA-DRB1*01:01-hACE-2 mice challenged with same SARS-CoV-2 VOCs (Supplementary Figure S4, right panel).The peptide binding IgG level was significantly higher for all nine "universal" B cell epitopes in COVID-19 patients (Supplementary Figure S4, left panel) and in vaccinated triple transgenic mice (Supplementary Figure S4 Altogether, these results indicate that immunization with the pan-variant SARS-CoV-2 vaccine bearing conserved "universal" Band T-cell epitope induced cross-protective antibodies, CD8 + and CD4 + T cells that infiltrated the lungs, facilitate virus clearance, and reduced COVID-19-related lung pathology following infection with various multiple SARS-CoV-2 VOCs.

Discussion
COVID-19 remains a serious threat with continued high rates of morbidity and mortality worldwide.The ongoing emergence of SARS-CoV-2 variants and sub-variants of concern, including the recent heavily mutated and highly transmissible Omicron subvariants, has led to vaccine breakthroughs that have contributed to prolonging the COVID-19 pandemic.Current Spike-based COVID-19 vaccines have made a substantial impact on the severity of the pandemic.Neutralizing antibody titers induced by current Spike-based vaccines are less effective against recent variants and sub-variants (41, 42), pointing to the urgent need to develop a next-generation B-and T-cell-based pan-variant SAS-CoV-2 vaccine-coronavirus vaccine that would be based not only on Spike protein but also on less-mutated non-Spike structural and non-structural antigens and epitopes.Such a universal CoV vaccine could induce broader and more durable protective immunity against infections and diseases caused by multiple emerging SARS-CoV-2 variants and sub-variants.
Much of the data on the efficacy of the current modified messenger RNA (mRNA) vaccines has shown that these vaccines elicited lower levels of neutralizing antibodies against newer SARS-CoV-2 variants than against the older variants (41).In the present report, we have identified "universal" CD8 + and CD4 + T-and B-cell epitopes conserved among all known SARS-CoV-2 variants, previous SARS and MERS coronavirus strains, and strains specific to different species that were reported to be hosts for SARS/MERS (bat, civet cat, pangolin, and camel).We used a combination of these highly conserved CD8+ and CD4+ T-and B-cell epitopes to design a multi-epitope pan-variant SARS-CoV-2 vaccine.The T-cell epitopes that constitute this pan-variant SARS-CoV-2 vaccine represent structural (Spike, Envelope, Membrane, and Nucleocapsid) and non-structural (orf1ab, ORF6, ORF7, ORF8, and ORF10) proteins.
We demonstrated that immunization of triple transgenic h-ACE-2-HLA-A2/DR mice with a pool of "universal" CD8 + T-cell, CD4 + T-cell, and B-cell peptides conferred protection against Washington, Alpha (B.1.1.7),Beta (B.1.351),Gamma (P.1), Delta (B.1.617.2), and Omicron (B.1.1.529)variants of SARS-CoV-2.The pan-variant SARS-CoV-2 vaccine was found to be safe, as no local or systemic side effects were observed in the vaccinated mice.Moreover, we found that the protection correlated with high frequencies of IFN-g CD4 + T cells, CD69 CD4 + T cells, IFN-g CD8+ T cells, and CD69 CD8+ T cells infuriating the lungs.We also found higher frequencies for the CD8 + T EM (CD44 + CD62L − ) cell population in the lungs of protected mice.High levels of peptidespecific IgG were also detected in protected animals, suggesting the contribution of Spike-specific antibodies in protection.A marked difference in the level of neutralizing viral titer was also observed between the vaccinated and mock-vaccinated groups of mice for all the studied variants.We observed no mortality in the vaccinated mice, irrespective of the SARS-CoV-2 variant.In contrast, high mortality was observed in the mock-vaccinated mice when challenged with six SARS-CoV-2 variants.The weight loss and survival shown in this study agree with previous reports in the context to Omicron B.1.1.529infection in the mouse models (43).Limited weight loss and less virus replication were reported for Omicron B.  (44).Both vaccines induced substantial spike-specific IFN-g-producing CD8 + and CD4 + T cells, which showed similar reactivity against Wuhan, Delta, and Omicron variants (44).In addition, their findings showed that central and effector memory Tcell subsets cross-reacted with Delta and Omicron variants (44).Similarly, in the present study, we showed a significant increase in the frequency of IFN-g-producing CD8 + and CD4 + T cells detected in the lungs of protected mice that received the pan-variant SARS-CoV-2 vaccine compared to non-protected mock-vaccinated mice.Overall, the vaccine was safe and immunogenic and provided crossprotection against multiple SARS-CoV-2 VOCs.
Interestingly, several earlier studies have performed epitope profiling in the existing COVID-19 mRNA vaccines (45,46).One such study has mapped immunogenic amino acid motifs and linear epitopes of primary sequence of SARS-CoV-2 spike protein that induce IgG in recipients of PfizerBioNTech COVID-19 mRNA vaccine (45).The obtained data identified various distinctive amino acid motifs recognized by vaccine-elicited IgG, a subset of those recognized by IgG from natural infection (hospitalized COVID-19 patients), which can mimic three-dimensional conformation (mimotopes).The identified dominant linear epitopes in the Cterminal region of the spike protein are identical with those of SARS-CoV, bat coronavirus, and epitopes that trigger IgG during natural infection, but have limited homology to spike protein of non-pathogenic human coronavirus (45).In another study, high-In this context, universal pan-variant vaccines generating Tand B-cell-mediated immunity like that of ours will be of crucial help.Such vaccines could induce strong humoral and cellular immunity against several viruses.On the one hand, B cells are activated upon recognizing antigens with B-cell receptors (BCRs) and further differentiate into memory B cells or plasma cells that can produce antigen-specific antibodies.Th2 cells help in B-cell activation by secreting cytokines such as IL 4 and IL-5.The induced neutralizing antibodies can block viral infection at the initial stage.On the other hand, antigens from the combined vaccine are taken up, processed, and presented to CD4 + and CD8 + T cells by antigenpresenting cells (APCs) through MHCII and MHCI antigen complexes, respectively.Then, CD4 + T cells are activated and can differentiate toward Th1, Th2, and memory CD4 + T cells.Th1 cells also promote the activation of CD8 + T cells by generating cytokines such as IFN-g, IFN-b, and IL-2.Activated CD8 + T cells can differentiate into effector and memory CD8 + T cells.The effector CD8 + T cells could kill and lyse infected cells by releasing cytokines, including IFN-g.Furthermore, the cross-reactive SARS-CoV-2-specific memory CD4 + and CD8 + T cells are present in up to 50% of unexposed, prepandemic, healthy individuals (UPPHI).However, the characteristics of cross-reactive memory CD4 + and CD8 + T cells associated with subsequent protection of asymptomatic COVID-19 patients (i.e., unvaccinated individuals who never develop any COVID-19 symptoms despite being infected with SARS-CoV-2) remains to be fully elucidated.Studies from our group and others have detected crossreactive CD4 + and CD8 + T cells, directed toward specific sets of conserved SARS-CoV-2 epitopes, not only from unvaccinated COVID-19 patients but also from a significant proportion of unexposed pre-pandemic healthy individuals (UPPHI) who were never exposed to SARS-CoV-2 (33,50,76,77).Cross-reactive SARS-CoV-2-specific memory CD4 + and CD8 + T cells are not only present in COVID-19 patients but also in up to 50% of UPPHI (50,(77)(78)(79)(80)(81)(82)(83).Moreover, pre-existing common cold coronavirus (CCCs)/SARS-CoV-2 cross-reactive memory CD4 + and CD8 + T cells are present in unvaccinated UPPHI who were never exposed to SARS-CoV-2 (33,50,76,77,(84)(85)(86)(87)(88)(89).These data suggest the presence of clones of memory CD4 + and CD8 + T cells in UPPHI induced following previous exposures with seasonal CCCs that cross-recognize conserved SARS-CoV-2 and CCCs epitopes (77,90,91).However, it is not yet known whether these cross-reactive memory CD4 + and CD8 + T cells: (i) preferentially cross-recognize the alpha CCCs (i.e., a-CCC-229E and a-CCC-NL63) or the beta CCCs (i.e., b-CCC-HKU1 and b-CCC-OC43); and (ii) the antigen-specificity, frequency, phenotype, and function of the cross-reactive memory CD4 + and CD8 + T cells associated with protection against COVID-19 severity in unvaccinated asymptomatic patients.Compared with unvaccinated severely ill COVID-19 patients and unvaccinated patients with fatal COVID-19 outcomes, unvaccinated asymptomatic COVID-19 patients displayed significantly (i) higher rate of the a-CCC strain 229E (a-CCC-229E); (ii) higher frequencies of functional memory CD134 + CD137 + CD4 + and CD134 + CD137 + CD8 + T cells directed toward cross-reactive a-CCCs/SARS-CoV-2 epitopes from structural, non-structural, and regulatory proteins; and (iii) lower frequencies of cross-reactive exhausted PD-1 + TIM3 + TIGIT + CTLA4 + CD4 + and PD-1 + TIM3 + TIGIT + CTLA4 + CD8 + T cells.These findings (i) support a crucial role of functional, poly-antigenic a-CCCs/SARS-CoV-2 crossreactive memory CD4 + and CD8 + T cells, induced following previous exposures to a-CCC strains, in protection against subsequent severe disease caused by SARS-CoV-2 infection; and (ii) provides a strong rationale for the development of broadly protective, T-cell-based, multi-antigen universal pan-coronavirus vaccines.
Since the B-and T-cell epitopes used in this study are highly conserved between SARS-CoV-1 and MERS-CoV, it is likely that protection will be observed against these strains as well.However, providing direct evidence of protection induced by our multiepitope vaccine against SARS-CoV-1 and MERS-CoV would require (i) generating a new DPP4/HLA-DR0101/HLA-A*0201 triple transgenic mouse model that needs backcrossing the DPP4 transgenic mice with our HLA-DR0101/HLA-A*0201 double transgenic mice, a process that is time consumable, as it will take several months to establish; and (ii) extensive in vivo and in vitro studies.Hence, demonstration of the breadth of protection induced by our multi-epitope vaccine against SARS-CoV-1 and MERS-CoV will be the subject of future independent reports in continuation to the existing study.
In conclusion, we report that a CoV vaccine targeting conserved B-and T-cell epitopes was safe, immunogenic, and provided crossprotection against six SARS-CoV-2 variants of concern, supporting the next-generation vaccine strategy.

Figure 1B ,
Figure1B, left panel) or Omicron (p < 0., Figure1B, right panel) variants.This observation was consistent regardless of whether the CD8 + T-cell targeted epitopes were from structural or nonstructural SARS-CoV-2 protein antigens, suggesting that strong CD8 + T-cell responses specific to selected "universal" SARS-CoV-2 epitopes were commonly associated with better COVID-19 outcomes.In contrast, low SARS-CoV-2-specific CD8 + T-cell responses were more commonly associated with severe onset of disease.Similarly, higher frequencies of functional IFN-g-producing CD4 + T cells ASYMP COVID-19 patients (mean SFCs > 25 per 1 × 10 6 pulmonary immune cells) were detected, irrespective of infection with Beta (p < 0.5, Figure1C, left panel) or Omicron (p < 0., Figure1C, right panel) variants, whereas reduced frequencies of IFN-g-producing CD4 + T cells were detected in SYMP COVID-19 patients, irrespective of infection with Beta (p < 0.5, Figure1C, left panel) or Omicron (p < 0., Figure1C, right panel) variants.This observation was consistent regardless of whether the CD4 + T-cell targeted epitopes were from structural or non-structural SARS-CoV-2 protein antigens.Our results suggest that strong CD4 + T-cell responses specific to selected "universal" SARS-CoV-2 epitopes were commonly associated with better COVID-19 outcomes.In contrast, low SARS-CoV-2-specific CD4 + T-cell responses were more commonly associated with severe disease onset.Taken together, these results (1) demonstrate an important role of SARS-CoV-2-specific CD4 + and CD8 + T cells directed against highly conserved structural and non-structural SARS-CoV-2 epitopes in protection from severe COVID-19 symptoms, (2) highlight the potential importance of these highly conserved "asymptomatic" epitopes in mounting protected CD4 + and CD8 + T cell responses against multiple SARS-CoV-2 VOCs, and (3) support targeting these conserved epitopes with a vaccine.

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FIGURE 3 Histopathology and immunohistochemistry of the lungs from in triple transgenic HLA-A*02:01/HLA-DRB1*01:01-hACE-2 mice vaccinated and mock-vaccinated mice.(A) Representative images of hematoxylin and eosin (H & E) staining of the lungs harvested on day 14 p.i. from vaccinated (left panels) and mock-vaccinated (right panels) mice.(B) Representative immunohistochemistry (IHC) sections of the lungs were harvested on Day 14 p.i. from vaccinated (left panels) and mock-vaccinated (right panels) mice and stained with SARS-CoV-2 Nucleocapsid antibody.Black arrows point to the antibody staining.Fluorescence microscopy images showing infiltration of CD8 + T cells (C) and of CD4 + T cells (D) in the lungs from vaccinated (left panels) and mock-vaccinated (right panels) mice.Lung sections were co-stained using DAPI (blue) and mAb specific to CD8 + T cells (pink) (magnification, ×20).The white arrows point to CD8 + and CD4 + T cells infiltrating the infected lungs.