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Background: Patients with primary and secondary immunodeficiencies have shown an impaired humoral immune response to COVID-19 vaccination. It is therefore of paramount importance to investigate anti-SARS-CoV-2 antibody levels in plasma pools and in immunoglobulin (IgG) products used to treat these patients. AIM: To assess the evolution of anti-SARS-CoV-2 antibodies (S protein) in plasma pools and IgG products and its neutralizing activity to original-type virus (Wuhan) and the variants of concern (VOC), including Omicron. Method(s): Healthy donors plasma pools collected in the US and Europe, and the subsequent intravenous (Flebogamma DIFand Gamunex-C, Grifols) and subcutaneous (Xembify, Grifols) IgG manufactured batches were followed from March 2020. Anti-SARS-CoV-2 S protein IgG titers were determined in plasma pools and in IgG batches by ELISA. Neutralization assays analyzed the capacity of IgG products to neutralize original-type virus and VOC (Alpha, Beta, Delta, Omicron BA.1 and BA.5), using pseudo viruses expressing S protein. Results were expressed as the dilution producing 50% neutralization (ID50). Result(s): In plasma pools, anti-SARS-CoV-2 S antibodies continuously increased throughout the study period regardless of the geographic origin. In the US, the first positive plasma pools were collected at the end of 2020. Since July 2021, an exponential increase over 30-fold of anti-SARS-CoV-2 S antibodies was reported. This trend continued increasing until the end of study period. Similarly, IgG products showed a similar evolution of anti-SARS-CoV-2 S antibodies. As expected, IgG batches released at the end of 2020 presented low SARS-CoV-2 neutralization activity. However, IgG products manufactured since August 2021 showed high neutralization activity against original-type virus and the rest of VOC. Regarding Omicron BA.5, a 5 to 10-fold increase was observed over time. Conclusion(s): This study reported the onset of elevated anti-SARS-CoV-2 antibody titers in plasma pools and IgG products since mid-2021, reflecting the evolution of the pandemic and vaccine campaigns. Intravenous and subcutaneous IgG products efficiently neutralized the current circulating VOC, Omicron BA.5. Further research is warranted to assess whether a clinical protective titer against SARS-CoV-2 and passive immunization is achieved in patients with immunodeficiencies treated with IgG products.Copyright © 2023 Elsevier Inc.
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Background: The pathogenetic mechanisms behind the development of long- COVID (LC) are largely unknown. Because both plasma SARS-CoV-2 RNAemia and dysregulated immunity have been correlated with COVID-19 severity, we evaluated whether they are associated with LC. Method(s): We consecutively enrolled unvaccinated hospitalized COVID-19 patients during acute-COVID-19 (T0) in March-October 2020 who either developed LC at a follow-up visit 2-3 months from virologic clearance (T1) or did not. LC was defined as persistence >=2 months after recovery of >=1 symptom: anosmia, dysgeusia, fever, gastrointestinal symptoms, dyspnoea, fatigue, musculoskeletal pain, muscle weakness, brain fog. We measured: SARS-CoV-2 RNAemia (RT-qPCR, log10(copies/mL)), magnitude (ELISA, AUC) and functionality (pseudovirus neutralization, ID50;Fc-mediated functions, %ADCC) of SARS-CoV-2-specific antibodies, SARS-CoV-2-specific B and CD4-T-cells (Immunophenotype, AIM and ICS assays). Result(s): We enrolled 48 COVID-19 individuals, 38/48 (79.2%) developed LC (LC+) and 10 did not (LC-). LC+ and LC- had similar co-morbidities and symptoms in the acute phase (Fig.1A), and the majority showed a radiologically documented SARS-CoV-2 pneumonia. The SARS-CoV-2 RNAemia did not differ between groups at both time points. The levels of RBD-specific Abs, as well as their functionality, appeared to increase over time in the LC- group but not in the LC+ (Fig.1B-D). Similarly, a trend towards increased RBD-specific B-cells was observed over time in the LC- group but not in LC+ (Fig.1E). B-cell immunophenotyping showed a significant increase over time of classical memory B cells (MBCs) at the expenses of activated MBCs (Fig.1F-G) as well as an IgA class-switching in the LC- group compared to LC+ (Fig.1H-I). Furthermore, LC+ showed a faster decline of SARS-CoV-2-specific (CD69+CD137+) CD4- TEMRA and CD4-TEM (Fig.1L-M). Finally, IFN-gamma-producing TREG of LC- individuals increased over time (Fig.1N). Conclusion(s): Acutely ill, hospitalized COVID-19 patients developing LC feature a dysregulated SARS-CoV-2-specific humoral as well as B- and T-cell response, in both magnitude and functionality, suggesting a link between dysregulated SARS-CoV-2-specific adaptive immunity and LC development. The fine understanding of the factors contributing to such dysregulation in LC patients is strongly needed, that might further inform targeted therapeutic interventions. (Figure Presented).
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Background: Although mRNA SARS-CoV-2 vaccines have received emergencyuse- authorization for infants age 6 months and older, vaccine uptake is slow, stressing that questions of safety and durability of vaccine efficacy remain prominent. Method(s): Infant rhesus macaques (RMs) (n=8/group) at 2 months of age, comparable to human toddler age, were immunized intramuscularly at weeks 0 and 4 with 30mug stabilized prefusion SARS-CoV-2 S-2P spike (S) protein (Washington strain) encoded by mRNA encapsulated in lipid nanoparticles (mRNA-LNP) or 15mug S protein mixed with 3M-052 in stable emulsion (Protein). At 1 year, vaccinated and age-matched unvaccinated RM (n=8) were challenged intranasally (106pfu) and intratracheally (2x106pfu) with B.1.617.2. Lung radiographs and pathology were blindly assessed, viral N gene RNA (vRNA) copies were measured by qPCR in pharyngeal swabs and lung, and neutralizing antibody and peripheral blood T cell responses were measured. Result(s): At 1 year, D614G-specific neutralizing antibody (nAb) titers were still detectable in the Protein (ID50=755;range: 359-1,949) and mRNA-LNP groups (ID50=73;range: 41-240). Both vaccines also induced cross-neutralizing antibodies to B.1.617.2. Peripheral blood CD4+ T cell responses to the ancestral spike protein at week 52 did not differ between the groups. However, median CD8+ T cell responses were higher (p=0.002, Mann Whitney) in the mRNA-LNP group (2.8%;range: 0.9%-7.1%) compared to the Protein group (0.8%;range: 0.1%-1.6%). Control RMs had significantly higher median vRNA copies/ml (1.4+/-2.7x108) in day 4 pharyngeal swabs compared to Protein (3.8+/-6.8x103) or mRNA-LNP (4.4+/-9.7x105) vaccinated RMs. Severe lung pathology was observed in 7 of 8 controls compared to 1 of 8 or 0 of 8 RMs in the mRNA-LNP or Protein group respectively. Protection against lung inflammation was associated with nAb titers (r=-0.592, p=0.003) (Figure 1). Conclusion(s): These results demonstrate that despite lower vaccine doses compared to adults, both protein and mRNA vaccines were safe, induced durable immune responses and provided comparable protective efficacy against infection with a heterologous SARS-CoV-2 variant in infants, implying that early life vaccination of human infants may lead to durable immunity. Neutralizing ID50 antibody titers are a correlate of protection in infant RMs challenged with SARS-CoV-2.
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Background. There are few data on immune correlation of protection from breakthrough Omicron (B.1.1.529) infection in individuals who received booster vaccines. We thus compared a neutralizing antibody titers against Omicron within the first month after the mRNA booster at the time before omicron wave between healthcare works (HCWs) who experienced Omicron breakthrough infections and HCWs without Omicron infections. Methods. We enrolled HCWs without the history of SARS-CoV-2 infection who agreed with blood sampling 2 weeks after booster vaccination at Asan Medical Center, Seoul, South Korea, between November 2021 and December 2022 (Delta dominant era). We identified breakthrough infections by performing SARS-CoV-2 RT-PCR though nasopharyngeal swab specimen in HCWs who had COVID-19-related symptoms or had known exposure to confirmed SARS-CoV-2-infected patients, between 1 February and 25 April 2022 (Omicron dominant era). SARS-CoV-2 S1-specific IgG antibody titers were measured using enzyme-linked immunosorbent assay (ELISA). Plasma levels of live-virus neutralizing antibodies were measured using a microneutralization assay with SARS-CoV-2 omicron variants. Results. Among 134 HCWs, 69 (52%) received two-dose ChAdOx1 nCoV-19 followed by BNT162b2, 50 (37%) three-dose BNT162b2, and 15 (11%) 3-dose mRNA-1273. Of them, 57 (43%) experienced breakthrough Omicron infection at median 121 days (IQR 99-147) after booster vaccination (breakthrough group), and the remaining 77 (57%) did not experience Omicron infection (non-breakthrough group). There was no significant different in 'peak' SARS-CoV-2 S1-specific IgG level between breakthrough group (median 4484.4 IU/mL) and non-breakthrough group (median 4194.9 IU/mL, p value=0.39). In addition, there was no significant difference in 'peak' neutralizing antibody titer (ID50) against Omicron between breakthrough group (median 2597.9) and non-breakthrough group (median 2597.9, p value=0.86). (Table Presented) Serum samples were obtained from 134 healthcare workers 2 weeks after booster vaccination. Samples were analysed for SARS-CoV-2 S1-specific IgG antibody titers using enzyme-linked immunosorbent assay (ELISA) and plasma levels of live-virus neutralizing antibodies using a microneutralization assay with SARS-CoV-2 omicron variants. There was no significant difference in 'peak' SARS-CoV-2 S1-specific IgG level (A) and 'peak' neutralizing antibody titer (ID50) against Omicron (B) between breakthrough group and non-breakthrough group. Conclusion. We did not find the correlation of neutralizing antibody titers about several months before infection with breakthrough Omicron infections. These data suggest rapidlywaning neutralizing titers to protect mild illnesses or asymptomaticOmicron infections several months after current booster COVID-19 vaccination in HCWs.
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Background: Heterologous prime-boost SARS-CoV-2 vaccination is a widely accepted strategy during the COVID-19 pandemic, which generated a superior immune response than homologous vaccination strategy. Objective: To describe immunogenicity of heterologous prime-boost vaccination with inactivated vaccine, CoronaVac, followed by BNT162b2 and 5-month booster dose with BNT162b2 in healthy Thai adolescents. Methods: Adolescents aged 12-18 years were randomized 1:1:1:1 to receive CoronaVac (SV) followed by BNT162b2 (PZ) 30 or 20 µg at either 3- or 6-week interval (SV3w/PZ30µg, SV3w/PZ20µg, SV6w/PZ30µg or SV6w/PZ20µg). During the Omicron-predominant period, participants were offered a BNT162b2 booster dose 30, 15, or 10 µg. Immunogenicity was determined using IgG antibody against spike-receptor-binding domain of wild type(anti-S-RBD IgG) and surrogate virus neutralization test(sVNT) against Delta variant at 14 days and 5 months after the 2nd dose. Neutralization tests(sVNT and pseudovirus neutralization test; pVNT) against Omicron strain were tested pre- and 14 days post-booster dose. Results: In October 2021, 76 adolescents with a median age of 14.3 years (IQR 12.7-16.0) were enrolled: 20 in SV3w/PZ30µg; 17 in SV3w/PZ20µg; 20 in SV6w/PZ30µg; 19 in SV6w/PZ20µg. At day 14, the geometric mean(GM) of anti-S-RBD IgG in SV3w/PZ30µg was 4713 (95 %CI 4127-5382) binding-antibody unit (BAU)/ml, while geometric mean ratio(GMR) was 1.28 (1.09-1.51) in SV6w/PZ30µg. The GMs of sVNT against Delta variants at day 14 among participants in SV3w/PZ30µg and SV6wk/PZ30µg arm were 95.3 % and 99.7 %inhibition, respectively. At 5 months, GMs of sVNT against Delta variants in SV3w/PZ30µg were significantly declined to 47.8 % but remained at 89.0 % inhibition among SV6w/PZ30µg arm. In April 2022, 52 adolescents received a BNT162b2 booster dose. Proportion of participants with sVNT against Omicron strain > 80 %inhibition was significantly increased from 3.8 % pre-booster to 67 % post-booster. Proportion of participants with pVNT ID50 > 185 was 42 % at 14 days post 2nd dose and 88 % post booster, respectively. Conclusions: Heterologous prime-boost vaccination with CoronaVac followed by BNT162b2 induced high neutralizing titer against SARS-CoV-2 Delta strain. After 5-month interval, booster with BNT162b2 induced high neutralizing titer against Omicron strain.Thai Clinical Trials Registry (thaiclinicaltrials.org): TCTR20210923012.
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Objective: To examine the temporal trends of humoral and cell-mediated immune responses to SARS-CoV-2 mRNA vaccines among multiple sclerosis (MS) patients on different immunomodulatory therapies. Background: The impact of various MS medications on the immune responses to SARS-CoV-2 vaccine is of acute interest to patients and clinicians. Design/Methods: 22 MS patients treated with ocrelizumab (OCR, n=9), natalizumab (NTZ, n=8), fumarates (FUM, n=5;diroximel fumarate, 3 and dimethyl fumarate, 2) received BNT162b2 (Pfizer, n=15) or mRNA-1273 (Moderna, n=7) vaccines. Blood samples were collected before and after each of the two vaccine doses, and 2 months after second vaccine dose. AntiSARS-CoV-2 spike protein titers were measured using quantitative assay (Labcorp). Antibody neutralization was measured with a lentivirus-based pseudovirus particle expressing the D614 spike protein (Labcorp-Monogram Biosciences). T-cell reactivity was determined by measuring interferon-gamma and interleukin-2 in response to stimulation with SARS-CoV-2 peptides. Results: All patients in NTZ and FUM cohorts, but only 22% (2/9) of OCR cohort developed anti-spike and neutralizing antibodies. The highest titers were measured after the second vaccine dose, without significant difference between the NTZ and FUM cohorts in anti-spike IgG (69.7+/-55.1 vs 56.0+/-36.7 arbitrary units/ml) or neutralizing ID50 (1513+/-1317 vs 942+/ -566). Two months after the second vaccine, the antibody titers and neutralizing ID50 decreased by 72% and 79% in NTZ cohort, respectively, and by 45% and 49% in FUM cohort. T-cell reactivity was observed in all cohorts as early as 7 days after the first vaccine, and further increased following the second vaccine. Conclusions: Patients on NTZ and FUM mounted robust antibody responses to SARS-CoV-2 mRNA vaccines, in contrast to OCR-treated patients. T-cell responses were comparable among all three treatment cohorts. Two months after the second vaccine, the serological responses decreased by 45-79%. These findings may inform the optimal timing of additional vaccine doses for MS patients.
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Background: The emergence of new SARS-CoV-2 variants raises concerns whether preexisting artificial (vaccine-induced) and natural immunity from prior COVID-19 prevents re-infections. Here, we investigated the differences in primary humoral immune response following SARS-CoV-2 variants of concern (VOCs) infection and aimed to identify the key mutations involved in these differences. Methods: Patients with primary PCR-proven SARS-CoV-2 infection with no history of previous COVID-19 vaccination were included between October 2020 and May 2021 at Amsterdam UMC and via the Dutch SARS-CoV-2 sequence surveillance program. Serum was collected 4-8 weeks after symptom onset and tested for IgG binding and pseudovirus neutralization of the wild-type (WT, Wuhan/D614G), Alpha, Beta and Delta variants. Results: We included 51 COVID-19 patients, who were infected with the WT (n=20), Alpha (n=10), Beta (n=9) or Delta variant (n=12). Generally, the highest neutralization titers were against the autologous virus. After stratifying for hospitalization status, non-hospitalized patients infected with the WT (ID50 817) or Alpha (ID50 2524) variant showed the strongest geometric mean autologous neutralization, followed by the Delta variant (ID50 704) infected participants. By contrast, only one participant infected with the Beta variant showed strong autologous neutralization (median ID50 171). The VOCs also differed in their ability to induce cross-neutralizing responses, with WT-infected patients showing the broadest immune response, followed by Alpha, Delta and Beta infected participants. Additionally, participants infected with the WT, Alpha or Delta variant showed the lowest cross-neutralization against the Beta variant, with a median 5.0-fold (2 to 16-fold), 7.7-fold (2 to 32-fold), and 5.3-fold (1 to 19-fold) reduction compared to the autologous neutralization, respectively. We identified the E484K mutation as the key mutation responsible for this low cross-neutralization. Conclusion: We demonstrated that even small differences in the S protein influences the polyclonal antibody response following infection. The low level of (cross-)neutralization induced by the Beta variant may implicate a higher re-infection risk, but further research of the memory B cell compartment and clinical studies are needed. The broadest cross-neutralizing response observed for WT-infected patients suggests that artificial immunity induced by the current approved COVID-19 vaccines already protects against many re-infections.
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Background: Critical COVID-19 occurs ca. 7d from symptoms onset, and is associated to immune dysregulation as well as SARS-CoV-2 detection in plasma (i.e. viremia). We hereby sought to detail the association between SARS-CoV-2 viremia measured at the end of the first week of disease and immune phenotypes/function in COVID-19 patients. Methods: We consecutively enrolled patients hospitalized in the acute phase of ascertained SARS-CoV-2 pneumonia. In this disease stage, we studied SARS-CoV-2 viremia (RT-PCR) and cytokines (MACSPlex), HLA-DR+CD38+ activated, GRZB+PRF+ pro-cytolitic T-cells, intracellular cytokine production (IL-2, IFNγ, TNFα, IL-4, IL-17A) after SARS-CoV-2 challenge (S-N-M-peptide pool). Simultaneous Th1-cytokines production (polyfunctionality) and amount (iMFI) was assessed. Humoral response: anti-S1/S2 IgG, anti-RBD total-Ig, IgM, IgA, IgG1 and IgG3 (ELISA), pseudoviruses neutralization (ID50) and Fc-mediated functions (%ADCC). Results: Out of 54 patients, 27 had detectable viremia (V+). Albeit comparable age and co-morbidities, V+ patients more frequently required non-invasive/invasive ventilatory support (p=0.035), with a trend to higher death (p=0.099) vs patients with undetectable viremia (V-)(Fig.1A). V+ displayed higher circulating IFN-α (p=0.002) and IL-6 (0.003), lower activated HLA-DR+CD38+CD4 (p=0.01) and CD8 (p=0.02), with no differences in GRZB+PRF+ T-cells. V+ featured reduced SARS-CoV-2-specific cytokine-producing T-cells, reaching significance for IFNγ+CD4 (p=0.02), TNFα+CD8, IL-4+CD8 (p=0.04) (Fig.1B-C), with lower bi-and tri-functional SARS-CoV-2-specific CD4 Th1, reaching significance for IL-2+TNFα+CD4 (p=0.03) (Fig.1D). A trend towards lower cytokines iMFI in bi-and tri-functional SARS-CoV-2-specific CD4 Th1 was observed in V+, reaching significance for IL-2+TNFα+CD4, p=0.004. V+ displayed lower anti-S IgG, anti-RBD total-Ig, IgM, IgG1 and IgG3 (Fig.1E), with lower ID50 and %ADCC vs V-(Fig.1F-G). Conclusion: Hospitalized COVID-19 patients with detectable plasma SARS-CoV-2 RNA in the acute phase of disease present worse outcome, higher inflammatory cytokines, fewer activated and SARS-CoV-2-specific polyfunctional T-cells, suggesting a link between SARS-CoV-2 viremia at the end of the first stage of disease and immune dysregulation. Whether high ab initium viral burden and/or intrinsic host factors contribute to a delayed and/or exhausted immune response in severe COVID-19 remains to be elucidated, to further inform strategies of targeted therapeutic interventions.
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Background. The zoonotic emergence of SARS-CoV-2 quickly developed into a global pandemic. Multiple vaccine platforms have been advanced to clinical trials and emergency use authorization. The recent emergence of SARS-CoV-2 virus variants with Spike receptor-binding domain (RBD) and N-terminal domain (NTD) mutations, highlights the need for next-generation vaccines that can elicit immune responses that are resilient against Spike mutations. Methods. Using a structure-based vaccine design approach, we developed multiple optimized SARS-CoV-2 nanoparticle immunogens that recapitulate the structural and antigenic profile of the SARS-CoV-2 prefusion spike. We assessed these immunogens in murine immunogenicity studies and in a K18-hACE2 transgenic mouse model with a SARS-CoV-2 challenge. Immune sera from vaccinated mice were assessed for SARS-CoV-2 binding, and neutralization against SARS-CoV-2, variants of concern, and the heterologous SARS-CoV-1 virus. Results. In combination with a liposomal-saponin based adjuvant (ALFQ), these immunogens induced robust binding, ACE2-inhibition, and authentic virus and pseudovirus neutralization. A Spike-Ferritin nanoparticle (SpFN) vaccine elicited neutralizing ID50 titers >10,000 after a single immunization, while RBD-Ferritin (RFN) nanoparticle immunogens elicited ID50 titer values >10,000 values after two immunizations. Purified antibody from SpFN- or RFN-immunized mice was transfused into K18-ACE2 transgenic mice and challenged with a high-dose SARS-CoV-2 virus stock. In order to understand the breadth of vaccine-elicited antibody responses, we analyzed SpFN- and RBD-FN-immunized animal sera against a set of heterologous SARSCoV-2 RBD variants and SARS-CoV RBD. High binding titers with ACE2-blocking activity were observed against SARS-CoV-2 variants and the heterologous SARSCoV-1 RBD. Furthermore, both SpFN- and RFN-immunized animal sera showed SARS-CoV-1 neutralizing ID50 titers of >2000. Conclusion. These observations highlight the importance of SARS-CoV-2 neutralizing antibody levels in providing protection against emerging SARS-like coronaviruses and provide a robust platform for pandemic preparedness. Structure-based design enables development of a SARS-CoV-2 nanoparticle immunogen.
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Background. Global surveillance has identified emerging SARS-CoV-2 variants of concern (VOC) associated with increased transmissibility, disease severity, and resistance to neutralization by current vaccines under emergency use authorization (EUA). Here we assessed cross-immune responses of INO-4800 vaccinated subjects against SARS-CoV-2 VOCs. Methods. We used a SARS-CoV-2 IgG ELISA and a pseudo neutralization assay to assess humoral responses, and an IFNγ ELISpot to measure cellular responses against SARS-CoV-2 VOC in subjects immunized with the DNA vaccine, INO-4800. Results. IgG binding titers were not impacted between wild-type (WT) and B.1.1.7 or B.1.351 variants. An average 1.9-fold reduction was observed for the P.1 variant in subjects tested at week 8 after receiving two doses of INO-4800 (Figure 1a). We performed a SARS-CoV-2 pseudovirus neutralization assay using sera collected from 13 subjects two weeks after administration of a third dose of either 0.5 mg, 1 mg, or 2 mg of INO-4800. Neutralization was detected against WT and the emerging variants in all samples tested. The mean ID50 titers for the WT, B.1.1.7, B.1.351 and P.1. were 643 (range: 70-729), 295 (range: 46-886), 105 (range: 25-309), and 664 (range: 25-2087), respectively. Compared to WT, there was a 2.1 and 6.9-fold reduction for B.1.1.7 and B.1.351, respectively, while there was no difference between WT and the P.1 variant (Figure 1b). Next, we compared cellular immune responses to WT and SARS-CoV-2 Spike variants elicited by INO-4800 vaccination. We observed similar cellular responses to WT (median = 82.2 IQR = 58.9-205.3), B.1.1.7 (79.4, IQR = 38.9- 179.7), B.1.351 (80, IQR = 40.0-208.6) and P.1 (78.3, IQR = 53.1-177.8) Spike peptides (Figure 2). Conclusion. INO-4800 vaccination induced neutralizing antibodies against all variants tested, with reduced levels detected against B.1.351. IFNγ T cell responses were fully maintained against all variants tested.
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Background. First-generation COVID-19 vaccines are matched to spike protein of the Wuhan-H1 (WT) strain. Convalescent and vaccinee samples show reduced neutralization of SARS-CoV-2 variants of concern (VOC). Next generation DNA vaccines could be matched to single variants or synthetically designed for broader coverage of multiple VOCs. Methods. The synthetic consensus (SynCon®) sequence for INO-4802 SARSCoV-2 spike with focused RBD changes and dual proline mutations was codon-optimized (Figure 1). Sequences for wild-type (pWT) and B.1.351 (pB.1.351) were similarly optimized. Immunogenicity was evaluated in BALB/c mice. Pre-clinical efficacy was assessed in the Syrian Hamster model. Figure 1. Design Strategy for INO-4802 Results. INO-4802 induced potent neutralizing antibody responses against WT, B.1.1.7, P.1, and B.1.351 VOC in a murine model. pWT vaccinated animals showed a 3-fold reduction in mean neutralizing ID50 for the B.1.351 pseudotyped virus. INO-4802 immunized animals had significantly higher (p = 0.0408) neutralizing capacity (mean ID50 816.16). ID50 of pB.1.351 serum was reduced 7-fold for B.1.1.7 and significantly lower (p = 0.0068) than INO-4802 (317.44). INO-4802 neutralized WT (548.28) comparable to pWT. INO-4802 also neutralized P.1 (1026.6) (Figure 2). pWT, pB.1.351 or INO-4802 induced similar T-cell responses against all variants. INO-4802 skewed towards a TH1-response. All hamsters vaccinated with INO-4802 or pB.1.351 were protected from weight loss after B.1.351 live virus challenge. 4/6 pWT immunized hamsters were completely protected. pWT immunized hamsters neutralized WT (1090) but not B.1.351 (39.16). INO-4802 neutralized both WT (672.2) and B.1.351 (1121) (Figure 3). We observed higher increase of binding titers following heterologous boost with INO-4802 (3.6 - 4.4 log2-fold change) than homologous boost with pWT (2.0 - 2.4 log2 fold change) (Figure 4). Conclusion. Vaccines matching single VOCs, like pB.1.351 and pWT, elicit responses against the matched antigen but have reduced cross-reactivity. Presenting a pan-SARS-CoV-2 approach, INO-4802 may offer substantial advantages in terms of cross-strain protection, reduced susceptibility to escape mutants and non-restricted geographical use.