Toward a pan-SARS-CoV-2 vaccine targeting conserved epitopes on spike and non-spike proteins for potent, broad and durable immune responses

Background The SARS-CoV-2 non-Spike (S) structural protein targets on nucleocapsid (N), membrane (M) and envelope (E), critical in the host cell interferon response and memory T-cell immunity, are grossly overlooked in COVID vaccine development. The current Spike-only vaccines bear an intrinsic shortfall for promotion of a fuller T cell immunity. Vaccines designed to target conserved epitopes could elicit strong cellular immune responses that would synergize with B cell responses and lead to long-term vaccine success. We pursue a universal (pan-SARS-CoV-2) vaccine against Delta, Omicrons and ever-emergent new mutants. Methods and findings We explored booster immunogenicity of UB-612, a multitope-vaccine that contains S1-RBD-sFc protein and sequence-conserved promiscuous Th and CTL epitope peptides on the Sarbecovirus N, M and S2 proteins. To a subpopulation (N = 1,478) of infection-free participants (aged 18–85 years) involved in a two-dose Phase-2 trial, a UB-612 booster (third dose) was administered 6–8 months after the second dose. The immunogenicity was evaluated at 14 days post-booster with overall safety monitored until the end of study. The booster induced high viral-neutralizing antibodies against live Wuhan WT (VNT50, 1,711) and Delta (VNT50, 1,282); and against pseudovirus WT (pVNT50, 11,167) vs. Omicron BA.1/BA.2/BA.5 variants (pVNT50, 2,314/1,890/854), respectively. The lower primary neutralizing antibodies in the elderly were uplifted upon boosting to approximately the same high level in young adults. UB-612 also induced potent, durable Th1-oriented (IFN-γ+-) responses (peak/pre-boost/post-boost SFU/106 PBMCs, 374/261/444) along with robust presence of cytotoxic CD8+ T cells (peak/pre-boost/post-boost CD107a+-Granzyme B+, 3.6%/1.8%/1.8%). This UB-612 booster vaccination is safe and well tolerated without SAEs. Conclusions By targeting conserved epitopes on viral S2, M and N proteins, UB-612 could provide potent, broad and long-lasting B-cell and T-cell memory immunity and offers the potential as a universal vaccine to fend off Omicrons and new VoCs without resorting to Omicron-specific immunogens. Trial registration ClinicalTrials.gov ID: NCT04773067; ClinicalTrials.gov ID: NCT05293665; ClinicalTrials.gov ID: NCT05541861.

Regardless of vaccination status or hybrid immunity, each reinfection would add risks of mortality, hospitalization and other health hazards including burden of long COVID [24]. The long COVID associated with Omicron [25][26][27][28] and pre-Omicron VoCs [26] has loomed large from infections that didn't require hospitalization [28]. Immunization with current EUA approved vaccines could present only limited benefits to relieve long COVID [29,30].
While development of composition-updated (variant-specific) vaccines has been strongly advocated [38,39], a better strategy of "universal coronavirus vaccines" would be more urgently needed [40] for robust, broad, and durable immunity. The currently authorized Spike-only vaccines do not incorporate SARS-CoV-2's non-Spike structure proteins of envelope (E), membrane (M) and nucleocapsid (N), the regions critically involved in the host cell interferon response and T-cell memory [41][42][43]. Oversight of non-Spike proteins as targets could lead to an intrinsic shortfall for promotion of a fuller T cell immunity. Viral mutations are also known to occur in E, M and N (Table 1) [15][16][17][18][44][45][46][47], the structure proteins that are beyond recognition by current EUA-approved vaccines.

Design of Phase-2 extension booster trial and oversight
Booster 3 rd -dose following the Phase-2 trial primary 2-dose series. We conducted a booster vaccination study (n = 1,478) which was an extension arm of the Phase-2, placebocontrolled, randomized, observer-blind, multi-center primary 2-dose study (S1A Fig) [ClinicalTrials.gov ID: NCT04773067] in Taiwan with 3,844 healthy male or female adults aged >18 to 85 years (S1B Fig) who received two intramuscular doses (28 days apart) of 100 μg UB-612 or saline placebo. The objectives of the third-dose extension study were to determine the booster-induced safety and immunogenicity after unblinding, 6 to 8 months after the second dose.
The Principal Investigators at the study sites agreed to conduct the study according to the specifics of the study protocol and the principles of Good Clinical Practice (GCP); and all the investigators assured accuracy and completeness of the data and analyses presented. The protocol was approved by the ethics committee at the sites and all participants provided written informed consent. Full details of the booster trial design, inclusion and exclusion criteria, conduct, oversight, and statistical analysis plan are available in the study protocol.

Trial procedures of safety and immunogenicity
Reactogenicity in the primary and booster series. The primary safety endpoints of the Phase-2 primary series (Days 1-365) and extension booster trial (recorded until 14 days postbooster and followed up study end) were to evaluate safety and tolerability. Vital signs were assessed before and after each injection. After each injection, participants had to record solicited local and systemic AEs in their self-evaluation e-diary for up to seven days while skin allergic reactions were recorded in their e-diary for up to fourteen days. Safety endpoints include unsolicited AEs reported for Days 1 to 57 in primary series and Days 1 to 14 in the booster phase. The overall safety was followed until the end of this study. Complete details for solicited reactions are provided in the study protocol.
Scope of immunogenicity investigation. The primary immunogenicity endpoints were the geometric mean titers (GMT) of neutralizing antibodies against SARS-CoV-2 wild-type

PLOS PATHOGENS
Heading toward a pan-SARS-CoV-2 vaccine The presence of T cell epitopes is critical for the induction of B and T cell memory responses against viral antigens. SARS-CoV-2 CTL and Th epitopes, validated by HLA binding and T cell functional assays, are highly conserved between SARS-CoV-2 and SARS-CoV-1 viruses, with minor between-variant differences seen only at S The secondary immunogenicity endpoints include anti-S1-RBD IgG antibody, inhibitory titers against ACE2:RBDWT interaction, and T-cell responses assayed by ELISpot and Intracellular Staining. The RBD IgG ELISA was fully validated using internal reference controls and results expressed in end-point titers. A panel of 20 human convalescent serum samples from hospitalized Taiwan COVID-19 patients aged 20 to 55 years were also tested for comparison with those in the vaccinees. Human peripheral blood mononuclear cells (PBMCs) were used for monitoring T cell responses (ELISpot and ICS). The constructs of the UB-612 vaccine product, all bioassay methods and statistics are detailed in the Supporting Information (S1-S8 Methods).

Booster trial population
After unblinding of Phase-2 trial, 1,478 of the 3,844 healthy study participants who completed the 2-dose primary vaccine series (28 days apart) of 100-μg UB-612 (S1A Fig) were enrolled to receive an additional 100-μg booster 3 rd -dose at 6 to 8 months after the second shot. The booster vaccinees were followed for 14 days to evaluate safety and immunogenicity. The vast majority of participants were of Taiwanese origin, with two groups aged 18-65 years (76%) and 65-85 years (24%) (S1B Fig).

Reactogenicity and safety
No vaccine-related serious adverse events (SAEs) were recorded; the most common solicited AEs were injection site pain and fatigue, mostly mild and transient (S2

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Heading toward a pan-SARS-CoV-2 vaccine primary series and homologous-boosting could induce pronounced Th1-predominant immunity.
Similar IFN-γ profiles were observed for those stimulated with Th/CTL peptide pool alone (Fig 1A), which increased from the baseline 1.3 to a high peak at 322 SFU/10 6 cells on Day 57 [48], maintained at 182 SFU/10 6 cells (~57%) at pre-boosting and remained strong at 317 SFU/10 6 cells 14 days post-booster. T cell responses persisted robustly (60-70% of the high peak at Day 57) long over 6-8 months.
These results indicate that UB-612 can induce a strong and durable IFN-γ + T cell immunity in the primary series, prompt a high level of memory recall upon boosting, and the fact that the presence of Th/CTL peptides is essential and principally responsible for the bulk of the T cell responses, while S1-RBD domain plays a minor role.

Robust CD4 + and CD8 + T cell activities by Intracellular Cytokine Staining (ICS)
Along with high levels of ELISpot-based T cell responses, ICS analyses revealed again substantial Th1-dominant %CD4 + T cells producing IFN-γ and IL-2, versus low level of IL-4 ( Fig 1B). Similar robust pattern was notable for %CD8 + T cells producing IFN-γ and IL-2 ( Fig 1C).
Vaccine recipients also showed cytotoxic T-cell responses, including CD8 + T cells expressing cytotoxic markers CD107a and Granzyme B ( Fig 1C) as observed in the primary series, accounting for a remarkable 3.6% of circulating CD8 + T cells after re-stimulation with S1-RBD + Th/CTL peptide pool, which persisted at a substantial 1.8% upon booster vaccination. Apparently, CD8 + T cell responses persisted robustly (50% of the high peak at Day 57) over 6-8 months as well. This suggests a potential of robust cytotoxic CD8 + T responses in favor of viral clearance once infection occurs.

Overview of B cell immunogenicity on antigenic and functional levels
Of the 871 Phase-2 study participants designated for Immunogenicity investigation, 302 participants had their serum samples collected at pre-boosting and 14 days post-booster for antigenic assay by anti-S1-RBD IgG ELISA, and functional assays by ACE2:RBD WT binding inhibition ELISA and by neutralization against live SARS-CoV-2 wild-type Wuhan strain (WT) by cell-based CPE method (S3 Fig). The results showed pronounced booster-induced increase of antibody titers that bound to RBD and inhibit/neutralize ACE2 interaction by respective 16-to 45-folds. These indicate that UB-612 booster vaccination could profoundly enhance both antigenic and functional activities.

Neutralizing antibodies against WT, Delta, Omicron BA.1 and BA.2
Functional blockade was further investigated comparatively on the occasion when Omicrons BA.1 and BA.2 dominate the pandemic scene. First, with limited available, affordable sources of viral variants, we investigated immune sera from 41 study participants across all age groups (18-65 years, n = 26; 65-85 years, n = 15). Neutralization measured using live virus, UB-612 booster elicited a neutralizing titer (VNT 50 ) against WT at 1,711 versus Delta variant at 1,282 (Fig 2A), representing a 1.3-fold reduction (GMFR, Geomean Fold Reduction). There was no significant age-dependent neutralization effect between young adults (18-65 yrs.) and the elderly (65-85 yrs.) with respect to either anti-WT or anti-Delta VNT 50 levels (Fig 2B), with a modest 1.2-to 1.7-fold GMFR of anti-Delta relative to anti-WT level.
As to Omicron BA.1 and BA.2 subvariants, when they sequentially dominated the pandemic scene, neutralization effects were measured by using pseudovirus for WT and Omicron subvariants. UB-612 booster elicited high neutralizing titers against WT at pVNT 50 of 6,245;

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Heading toward a pan-SARS-CoV-2 vaccine the elderly (65-85 years) with respect to either anti-WT or anti-Omicron pVNT 50 level ( Fig  2D and 2F). Both age groups showed a 5.0-to 7.6-fold reduction for anti-BA.2 relative to anti-WT. By all accounts compared with BA.1, booster vaccination exhibits only a minor 1.2-fold lower neutralizing activity against BA.2.
The inhibition of ACE2:RBD WT binding on ELISA correlates well with anti-WT ( Fig 4C) and anti-Delta VNT 50 (Fig 4D) findings, both showed a similar high correlative Spearman's rank correlation coefficients (r = 0.795 and 0.828, respectively). A lesser but significant correlation were also observed for ACE2:RBD WT binding inhibition and anti-Omicron BA.1 and anti-BA.2 pVNT 50 , with Spearman's correlation coefficients of r = 0.565 ( Fig 4E) and r = 0.602 (Fig 4F). A lower yet substantial correlation was also noted with anti-BA.5 pVNT 50 when only 12 sample points were available for regression analysis (Fig 4G).

High neutralizing-titer correlation between pseudovirus and live virus assays
The design of pseudovirus assay is based on contact with Spike protein only, while a live virus contains both Spike and non-Spike proteins that may behave differently in neutralizing strength. It is of high interest to address the issue as to whether an easier practice of pseudovirus assay could reflect the outcome from a live virus neutralization assay associated with UB-

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Heading toward a pan-SARS-CoV-2 vaccine 612 vaccination. It was found that the two viral-neutralizing titer assays against pseudo-virus (pVNT 50 ) and live-virus (VNT 50 ) are highly correlated, as exemplified by the assays against Wuhan wild-type virus (WT) with a Spearson r = 0.9517 ( Fig 5A) and that against Delta strain presents a Spearson r = 0.9041 (Fig 5B).

Discussion
The present Phase-2 UB-612 booster vaccination, proven safe and well tolerated without concerns of SAEs (S2 Fig), induces potent memory T cell immunity (Fig 1) that synergizes recalled B cell immunity with striking cross-neutralizing antibodies against WT, Delta and Omicrons (Figs 2 and 3). Of notable clinical interest, the booster uplifts a lower neutralizing antibody titer in the elderly [48] to a high level close to that in young adults regardless of viral mutant status (Figs 2 and 3). In addition, blockade of S1-RBD binding to ACE2 receptor correlates well with viral neutralization (Fig 4). Thus, the UB-612 vaccine platform, due to broadly recognizing conserved Th/CTL epitopes on Spike and non-Spike proteins, can maintain a target plasticity without much mutational distortion within the RBD domain of the target B immunogen. The present report reveals five salient findings.
Overall, UB-612 booster appears to perform on a par with or to bear a competitive edge over other vaccine platforms based on pseudovirus-neutralizing pVNT 50 , against Omicron BA.1/BA.2/BA.5. From a comparative view of between-vaccine platforms, the magnitude of viral-neutralizing strength would matter much more than a GMFR factor.
It should be noted that both pseudovirus pVNT 50 discussed above (Table 3) and live virus VNT 50 data to be discussed below (Table 4) have drawbacks as all assay methods by various vaccine platforms are not uniformly comparable. No standardized neutralization methods have been set to follow with. These data points are laid out for contrast, not for comparison purpose (with statistics). Nonetheless, a solid trend of platform-dependent difference in viralneutralization potency is discernable.

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Heading toward a pan-SARS-CoV-2 vaccine In addition, UB-612 booster presents a substantially high anti-BA.2 live-virus titer VNT 50 at 485, which is even far greater than the anti-BA.1 titers observed with other vaccines. In light of the true measure for neutralizing activity, the live virus assay would reflect better than the pseudovirus assay, as the former stands for the combined anti-viral activity against both Spike and non-Spike proteins, while the latter assay measures the strength against the Spike only.
Collectively, UB-612 booster performs on a par with or bears a competitive edge over other vaccine platforms in viral-neutralization potency, either pseudovirus or live virus, against Delta and Omicron BA.1, BA.2, and potentially the currently dominating BA.5.
Thirdly, UB-612 booster uplifts a lower viral-neutralizing titer generally associated with the elderly to a level approximately the same as that in the young adults. No significant age-dependent neutralization effect is evident between young adults and the elderly with respect to humoral immune responses against WT/Delta/BA.1/BA.2/BA.5 (Figs 2B, 2D, 2F and 3B-3D). This is of high clinical significance as elderly people, due to a decline in pathogen immunity, do not respond to immune challenge as robustly as young adults and so have a reduction in vaccine efficacy [54]. Thus, UB-612 as a primer or booster has a potential benefit not only for the elderly but also for immunocompromised people in general.

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Heading toward a pan-SARS-CoV-2 vaccine The pronounced, broadly-neutralizing profiles illustrate one unique feature of UB-612, i.e., the serum neutralizing antibodies are directed solely at the critical receptor binding domain (RBD) that reacts with ACE2. In contrast with the currently authorized full-length S proteinbased vaccine platforms, UB-612's RBD-only design leaves little room in non-conserved sites of S protein for viral mutation to occur and so may result in less immune resistance.
Thus, booster vaccination can prompt recall of high levels of parallel anti-WT neutralizing VNT 50 (Fig 4A) and RBD-ACE2 binding inhibition antibodies (Fig 4B), and both functional events are durable over Day 57 and Day 220 with a substantial 42%/88% retainment at 6 months or longer after the second shot. This is consistent with the long-lasting anti-WT VNT 50 effect with a half-life of 187 days observed in the Phase-2 primary series, in which ã 50% retainment was observed at 6 months relative to the peak response [48]. Further, the finding that the UB-612 induced a 140-fold higher increase in blocking the RBD:ACE2 interaction than by human convalescent sera (HCS) (Fig 4B) suggests that most of the antibodies in HCS may bind allosterically to the viral spike (N-or C-terminal domain of the S) rather than orthosterically to the RBD sites, which may include non-neutralizing anti-S antibodies to cause unintended side effects or Antibody-Dependent Effect (ADE) event. This warrants further investigation including sera from re-infections and breakthrough infections from all vaccine platforms.
UB-612 booster appears to trigger far greater T cell responses than those produced by the current Spike-only mRNA (BNT162b2) and adeno-vectored (ChAdOx1) vaccines [59]: e.g., the pre-boost/post-boost level of SFU/10 6 cells (related to Delta strain) under homologous boosting for the 3-dose of ChAd/ChAd/ChAd were 38/45 and that of BNT/BNT/BNT were 28/82; and those under heterologous boosting were 42/123 for ChAd/ChAd/BNT and 36/108 for BNT/BNT/ChAd. For those currently licensed COVID vaccines, it is worthy to note that the fourth vaccine jab (the 2 nd booster) does not increase T cell response [60]: e.g., the 28 days post-3 rd dose/pre-4 th dose/14 days post-booster level of SFU/10 6 cells (related to Delta strain) for the 4-dose of ChAd/ChAd/BNT/BNT were 133/19/108 and that of BNT/BNT/BNT/BNT were 62/14/80.
The lackluster booster-recalled T cell immunity seen with mRNA and adeno-vectored vaccines [59,60] may reflect the dwindling, weakened B cell humoral responses and clinical efficacy. A booster 3 rd -dose of mRNA vaccines could compensate the waning immunity and reduce rates of hospitalization and severe disease, yet be less effective in protection against mild and asymptomatic infections [31][32][33][34][35][36]. At the time of Omicron BA.1 on the rise, vaccine effectiveness was seen reduced after booster (third dose) of mRNA vaccines in protection against COVID symptoms (45% at 10 weeks) [61] and hospital admission (55% at 12 weeks) [62].
In two retrospective large cohort studies, the elderly (aged ≧60) receiving the fourth dose of BNT162b2 (2 nd booster) while BA.2 infection was dominant also showed a modest and transient efficacy against severe disease (~60-75% protection, relative to the 1 st booster third dose) [63,64], and the effectiveness against infection completely wanes after 8 weeks [63].
Breakthrough infection could occur after the fourth dose [37], in particular amid the circulation of the dominant Omicron BA.5. The booster-compensated protection effectiveness offered by mRNA vaccines could be blunted soon upon boosting. Incessant, short-interval boosting with current mRNA vaccines could result in dwindling and weakened immune responses against Omicrons [65], for mechanisms remained to be elucidated.
While a substitute of the fourth dose with mRNA-1273 can elevate T cell response to a level of~240 SFU/10 6 cells [60], the increased response level appears to be lower than those by UB-612 at 261 SFU/10 6 cells pre-3 rd -dose boosting and at 444 SFU/10 6 cells 14 days post-booster (third dose) (Fig 1). The UB-612 vaccine, designed to target multiple conserved epitopes on both Spike and non-Spike proteins, could have underpinned the base for a fuller T cell immunity.
The potential clinical significance of a striking T-cell immunity elicited by UB-612 vaccine platform is supported by the development of a plain T-cell vaccine (CoVac-1) containing a sixpeptide backbone that, as a T-cell booster, triggered dramatic multifunctional CD4 and CD8 T-cell responses [66], which showed benefits to B-cell deficient, immunocompromised patients who could not mount B-cell antibody responses. The facts that potent memory CD4 and CD8 T cell memory can protect against SARS-CoV-2 infection in the absence of immune neutralizing antibodies [57,58,66] raises concerns over the fact that humoral antibody response has long been used as a sole bridging metric of protective immunity [67], which lacks full understanding of human post-vaccination immunity as antibody response is generally shorter-lived than virus-reactive T cells [68][69][70].
Further, the SARS-CoV-2's non-Spike structure E, M and N proteins are the regions critically involved in the host cell interferon response and T-cell memory [41][42][43]. These structural proteins of virus' main body when picked up by Antigen Presenting Cells (APC) and presented as viral peptides would fall beyond recognition by the currently authorized vaccines that are based on the outer spike protein only. Th1 cells help to stimulate B cells to make antibodies, and they can morph as well into memory helper CD4 + and cytotoxic CD8 + T cells to provide a long-lasting immune response [71]. UB-612's booster-enhanced broader, durable B and T cell immunity may make Omicron evasion less likely as the booster vaccination could behave closer to the breadth of natural, infection-induced immunity.
While neutralizing antibodies can block ACE2:RBD interaction and protect against initial infection, the non-Spike protein cross-reactive memory T cell immunity is essential for protection from severe disease and for long-term prevention against infection; and, as such, T cell immunity should be recognized as a measure for long-term vaccine success [72][73][74][75][76][77][78][79]. The role of T cells, in particular the recognition against non-Spike targets and the associated T cell responses, has long been underestimated and overlooked from the outset of COVID vaccine development.
The cross-reactive T cell responses can limit disease severity, reduce viral replication, and limit the duration of illness, and these potential durable immune responses revealed by UB-612 (Fig 1) would be a key component of a pan-SARS-CoV-2 vaccine. To what extent that vaccine booster-induced memory T cell immunity would contribute to vaccine effectiveness in the clinic against COVID-19 infection of any degree, as a leading actor or a supportive cast, has become a research subject of major clinical interest [80].
Of additional clinical interest with strong T cell immunity is its function of viral clearance. Persistent SARS-CoV-2 infections can contribute to long COVID as residual viable SARS-CoV-2 particles, viral replication, viral RNA and viral spike protein antigens could sustain in tissues of the convalescents [81][82][83]. As long COVID is found to be associated with a decline in IFN-γ-producing CD8 + T cell [84], enhancing T cell immunity for clearance of residual systemic infection (sustained viral reservoirs) could be a sensible strategy for prevention of long COVID.
Facing the dwindling vaccine effectiveness and emergence of viral variants with higher infectivity and immune evasion, development of composition-updated vaccines [38,39] or universal coronavirus vaccines [40] has been strongly advocated. To meet an urgent need and for a long-term fight against new mutants, one would look beyond the practice of frequent shortinterval booster jabs and resist clinging to use of variant-specific (e.g., omicron-updated) vaccines.
In fact, the recent bivalent vaccine mRNA-1273.214 (original wild-type Spike plus Omicron BA.1 Spike) as the fourth dose (second booster) was found to result in only 1.7-fold higher pseudovirus-neutralizing antibody titer (pVNT 50 ) against BA.5 as compared to that by the original mRNA-1273 [85]. The extra modest anti-BA.5 gain of pVNT 50 titer by the bivalent WT/BA.1 vaccine may not provide better protection efficacy against BA.5 infection.
Furthermore, bivalent WT/BA.5 mRNA vaccines (Pfizer and Moderna) at the 4 th dose in two studies have shown only 1.2 to 1.3-fold higher pVNT 50 neutralizing titer against BA.5 than the original wild type [86,87]. And, the bivalent booster-induced T cell response remained unchanged at a low level [87], relative to the original vaccine. These observations are in line with the mechanism of Immune Imprinting [88] that tips the bulk of antibodies to react with the first encounter wild-type strain or the initial vaccine type an individual exposed to, implicating also that variant-specific vaccine would not perform better than thought.
Of interest to note, to enhance vaccine immunity, a T cell vaccine BNT162b4 targeting conserved epitopes on non-Spike proteins, in combination with BNT162b5 BA.5-bivalent or BNT162 BA.1-bivalent vaccine, is being developed [ClinicalTrials.gov ID: NCT05541861]. The goal of the two vaccines in one shot is to deliver durable antibody and T-cell immune protection against severe disease and hospitalization for at least one year.
By and large, a pragmatic approach to curbing ever-emergent new mutants would be "universal (pan-Sarbecovirus) vaccines" targeting conserved nonmutable epitopes on Spike and non-Spike proteins of coronavirus. In that sense, a shift of Spike-only vaccine design to a paradigm by targeting conserved epitopes on both Spike and non-Spike proteins would be a workable option. To be competent for next-generation vaccines, conserved regions on non-Spike proteins (membrane and nucleocapsid) to serve as immunogens may also contribute to the development of pan-betacoronavirus vaccines [89].
By incorporation of five sequence-conserved Th/CTL epitope peptides [90] and a sixth idealized universal Th peptide which serves as catalyst in T cell activation [48], the UB-612-induced T cell immunity may enhance the clearance of the virally infected cells, regardless of Omicrons or future mutants, as their mutation sites are not to overlap any of the amino acid residues on the precision-designed S2, N, and M epitope peptides that are highly conserved (or rarely mutate) across all VoCs (Table 2). By design, UB-612 could provide strong memory T cell immunity that associates with potent, broadly-recognizing and durable live virus-neutralizing effect without resorting to Omicron-specific immunogens. Whether a strong, fuller and broadly-recognizing T cell immunity could help with prevention/minimization of long COVID warrants additional clinical research.
In summary, we have simultaneously characterized the booster-enhanced B-and T-cell immunity in a large (N = 1,378) Phase-2 study, demonstrating UB-612 can elicit a fuller T cell immunity that comprehensively recognize Spike (S1-RBD and S2) and non-Spike structure N and M proteins, which seeds the potential for viral clearance upon infection; and the induced B cell responses would broadly neutralize all VoCs regardless of varying mutational epitope locations. Our UB-612 multitope vaccine may serve as a universal vaccine primer and booster to ward off all VoCs and future mutants, for which a US-FDA approved CEPI supported largescale Phase-3 trial has also been underway to further evaluate the concept of protection efficacy. , and tested for neutralizing antibody levels that inhibit 50% of live SARS-CoV-2 wild-type, expressed as VNT 50 (WT, Wuhan strain) (functional), the inhibitory titers against S1-RBD binding to ACE2 by ELISA, expressed as μg/ mL (functional), and anti-S1-RBD IgG antibody titers by ELISA (antigenic). Statistical analysis was performed by the Student's t-test (ns, p>0.05; **** p<0.0001). General Hospital for their involvement in conducting the trial; and members of the IDMC for their dedication and guidance.

Supporting information
Special thanks are also extended to the clinical associates from StatPlus, Inc and UBI Asia; the CMC task forces from UBI Asia, United BioPharma, Inc. and UBI Pharma, Inc; team members at Institute of Biomedical Sciences, Academia Sinica for the live virus neutralization assay; and team members at the RNAi Core Facility, Academia Sinica for the pseudovirus neutralization assay. All health convalescent sera were supplied by Biobank at the National Health Research Institutes (NHRI), Taiwan.
Finally, thank Dr. Chuwan-chuen King for critical review of the manuscript; and colleagues from UBI Asia Hui-Kai Kuo, Wan-Yu Tsai, Han-Chen Chiu, Kuo-Liang Hou, Hope Liu, and Jennifer Cheng for their technical support and data collection. Special administrative support by Jalon Tai, Liang Kai Huang, Peter Hu and Fran Volz from the UBI group are also acknowledged with gratitude.