Humoral and functional <scp>SARS</scp> ‐ <scp>CoV</scp> ‐2‐specific T‐cell responses elicited in chronic lymphocytic leukaemia patients with <scp>GEO</scp> ‐ <scp>CM04S1</scp> a synthetic <scp>MVA</scp> ‐vectored <scp>COVID</scp> ‐19 vaccine

Chronic lymphocytic leukaemia (CLL) is characterized by immune dysfunction, increasing infection risk and reduced vaccine efficacy.1-3 CLL patients vaccinated against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) remain vulnerable to severe coronavirus disease 2019 (COVID-19) due to poor responses to mRNA vaccines.4, 5 Durable SARS-CoV-2 T-cell responses, which target a broad range of viral epitopes, are critical for viral clearance and less prone than antibodies to variant escape.6 COVID-19 vaccines that enhance both humoral and cellular immunity may offer improved protection and clinical outcomes for CLL patients. In haematopoietic stem cell transplant (HSCT) recipients, the Triplex multi-antigen cytomegalovirus vaccine using the modified vaccinia Ankara (MVA) platform proved safe and effective.7 Building on these results, we developed GEO-CM04S1, a synthetic MVA-vectored COVID-19 vaccine expressing both spike (S) and nucleocapsid (N) proteins of the ancestral Wuhan-Hu-1/D614G SARS-CoV-2 strain.8 In an ongoing randomized phase II trial in HSCT and Chimeric Antigen Receptor T-cell therapy (CAR-T)-cell patients, GEO-CM04S1 has elicited broad antibody responses against multiple SARS-CoV-2 variants and robust, long-lasting S- and N-specific T-cell responses.9 These findings supported a randomized Simon's two-stage trial (ClinicalTrials.gov: NCT05672355) comparing GEO-CM04S1 and BNT162b2 in CLL patients previously vaccinated with mRNA-based COVID-19 vaccines. Here, we report SARS-CoV-2-specific immune responses seen at interim analysis (the stage-one portion of Simon's two-stage trial design). Study design, methodology and statistical approach are detailed in Supporting Information S1. The primary outcome was to assess the proportion of participants with ≥3-fold rise in S- or N-specific interferon (IFN)-γ-secreting T cells from baseline to day 56 after the first vaccine booster (primary immune analysis, PIA). As per the Simon two-stage design, only the vaccine eliciting the defined PIA threshold in four or more participants (primary end-point) would be further assessed in stage-two of the trial. Secondary outcomes included safety and measuring levels of T-cell activation-induced markers (AIM) on S- and N-specific CD4+ and CD8+ T cells; S-, N- or receptor-binding domain (RBD)-specific Immunoglobulin G (IgG) titres and antibodies neutralizing SARS-CoV-2 pseudoviruses, based on ancestral Wuhan-Hu-1/D614G strain and variants of concern. Between July 2023 and July 2024, 31 consecutive patients met eligibility criteria (see Supporting Information S1) and were randomized to receive either the BNT162b2 (n = 15) or the GEO-CM04S1 (n = 16) vaccines. Of these, 29 participants were included in the intent-to-treat analysis and 27 in the PIA (Figure 1; Table S1). Patients were either under observation or had received CLL therapy (four participants in the BNT162b2 versus nine in the GEO-CM04S1 arm) before enrolment and all remained in remission throughout the 6-month study (Table S2). Most adverse events (AEs) were grade 1 and no grade ≥3 AEs occurred (Tables S3 and S4). COVID-19 was reported by four participants during the study (Table S4). Figure 2A illustrates the fold increase in S-, N- and M-specific IFN-γ secretion. In the GEO-CM04S1 arm, the geometric mean fold rise (GMFR) of S-specific IFN-γ secretion on day 56 was significantly higher than that of the BNT162b2 arm (GMFR 2.2 vs. 1.3). N- and M-specific IFN-γ secretion was similar between the two arms. Notably, 6 of 15 participants (40%) in the GEO-CM04S1 versus 2 of 14 (14.3%) in the BNT162b2 arm exhibited a ≥3-fold rise in S- or N-specific IFN-γ-secreting cells by day 56. Thus, only GEO-CM04S1 met the PIA (Table S5). Furthermore, in the GEO-CM04S1 arm, S-specific IFN-γ secretion was significantly more elevated than that in BNT162b2 patients also on days 28 (GMFR 1.8 vs. 1.1) and 84 (GMFR 1.9 vs. 0.84; Figure 2B). Among GEO-CM04S1 recipients with no prior CLL therapy (Figure S1A), GMFRs of S-specific IFN-γ secretion were significantly higher than those observed in BNT162b2 recipients on days 56 and 84 post-first booster. In patients exposed to treatment, such differences were not observed, though S-specific IFN-γ secretion in BNT162b2 trended higher than in GEO-CM04S1. SARS-CoV-2 specific IL-4 secretion remained minimal (data not shown). The GMFR difference between arms was significant for functionally activated AIM+ N-specific CD4+ T cells, approximately 10-fold higher in the GEO-CM04S1 (GMFR: 12.6, 19.6, 19.9) than in the BNT162b2 arm (GMFR: 1.7, 2.1, 1.3), on days 56, 112 and 180 post-first booster vaccination (Figure 2C). In contrast, differences in AIM+ S-specific CD4+ and CD8+ responses between the two arms were modest. As for N-specific CD8+ T-cell responses, GMFR was significantly higher in GEO-CM04S1 than in BNT162b2 arm on days 84 (5.7 vs. 1.0; p = 0.045, data not shown) and 112 (16.9 vs. 1.3). Among GEO-CM04S1 recipients without prior CLL therapy (Figure S1B,C), AIM+ N-specific CD4+ T-cell responses were significantly greater than those observed in BNT162b2 recipients. In patients who received prior therapy, responses trended higher in GEO-CM04S1 than in BNT162b2, and significance was reached on day 84 for N-specific CD8+. At baseline, no significant difference in S, N and RBD-binding IgG antibodies was observed between arms (Figure S2). The GMFR of S- and RBD-specific IgG generally trended higher in the BNT162b2 arm than in GEO-CM04S1 (Figure 2D). A statistically significant difference in RBD-specific IgG was observed only on day 28, with GMFRs of 2.1 in BNT162b2 versus 1.3 in GEO-CM04S1 (Figure S3A). In contrast, anti-N IgG levels remained significantly more elevated in the GEO-CM04S1 arm from day 56 through day 180 post-first booster, with GMFRs of 3.0, 2.6, 4.4 and 4.3, compared to 1.1, 0.9, 0.8 and 1.0 in the BNT162b2 arm. Although the fold rise of neutralizing antibodies (NAb) against Wuhan-Hu-1/D614G ancestral strain and Omicron BA.1 and XBB.1.5 variants were often higher in the BNT162b2 than in GEO-CM04S1 arm, the GMFR between arms was not statistically significant, except on day 180 post-first booster for Wuhan-Hu-1/D614G NAb (GMFR: 1.4 in BNT162b2 vs. 0.5 in GEO-CM04S1; Figure 2E; Figure S3B). Spearman's analysis showed a correlation between S-specific AIM+ T cells and IFN-γ in both arms, with minimal association with IgG levels. In the GEO-CM04S1 arm, N-specific IFN-γ and IgG were significantly correlated, whereas BNT162b2 showed correlation only between N-specific cellular assays (Table S6). GEO-CM04S1 met the per protocol primary design criterion of safely potentiating the cellular immune response when used as a booster vaccination in CLL patients. Only patients in the GEO-CM04S1 trial arm reached the predefined stage-one primary end-point for S- or N-specific IFN-γ-secreting cells (Figure 2A; Table S5). This novel vaccine is currently being further assessed in the ongoing stage-two portion of the trial. MVA, the viral vector of GEO-CM04S1 has intrinsic adjuvant properties and elicits humoral and cellular immune responses, even under post-transplant immunosuppression. MVA's property to preferentially infect professional antigen-presenting cells triggers strong innate and adaptive immune responses.7 These properties likely contributed to the GEO-CM04S1 improved booster capacity in CLL patients, including inducing enhanced SARS-CoV-2 specific IFN-γ, critical for infection resolution.10 Furthermore, AIM analyses assessing the T-cell breadth11 showed durable, significantly more robust N-specific T-cell activation in the GEO-CM04S1 versus Bnt162b2 arm (Figure 2C). This functionally activated N-specific immune response suggests that GEO-CM04S1 induces superior S- and N-specific cellular immunity. Although the role of N-specific responses in protection against SARS-CoV-2 remains incompletely defined, naturally acquired cellular responses targeting N and S are dominant and long-lasting in individuals recovering from COVID-19.12 An exploratory post hoc analysis indicated that CLL treatment-naive patients receiving GEO-CM04S1 had higher S-specific IFN-γ and AIM+ N-specific CD4+ T-cell responses than those given BNT162b2 (Figure S1). These findings suggest that GEO-CM04S1 may elicit an enhanced SARS-CoV-2-specific cellular immune response in this subgroup (Table S2).13 The humoral response for S- and RBD-IgG-binding antibodies and NAb showed GMFR trending higher for the BNT162b2 arm, with significance only at one time point for both IgG and ancestral Wuhan-Hu-1/D614G-specific NAb. In contrast, starting on day 56 post-first booster, the GEO-CM04S1 arm had a consistently greater fold rise than the BNT162b2 arm for N-IgG-binding antibodies (Figure 2D; Figure S3). Consequently, in CLL participants, the combination of S and N antigens in a single vector as in GEO-CM04S1 may increase the humoral response against SARS-CoV-2. The presence of dysfunctional clonal B cells in CLL contributes to a broad-spectrum hypogammaglobulinaemia, characterized by impaired immunoglobulin production. This defect leads to a marked reduction in SARS-CoV-2–NAb titres, which compromises the immunogenicity and clinical efficacy of COVID-19 vaccines in this patient population.3 Recent studies demonstrate that N-specific non-neutralizing antibodies (non-NAbs) can mediate Fc receptor-dependent effector functions, including antibody-dependent cellular cytotoxicity, contributing to viral control.14 Interestingly, while showing expected cellular assay associations,15 a correlation matrix uncovered a surprisingly strong N-specific IgG IFN-γ correlation exclusively in the GEO-CM04S1 arm (Table S6). Collectively, these findings support the use of GEO-CM04S1 vaccine for CLL patients as the inclusion of the N antigen may broaden protective immunity through non-NAb mechanisms. This study has limitations, including a small sample size and the absence of high-risk patients requiring active CLL therapy. Future research should investigate baseline IgG levels and document intravenous immunoglobulin usage to better assess vaccine response and variability. Larger cohorts are needed to clarify whether the lower Omicron BA.1- and XBB.1.5-specific NAb trends in GEO-CM04S1 recipients are clinically significant (Figure S3B). Nonetheless, our findings highlight a stronger cellular immune response to both S and N antigens in high-risk CLL patients boosted with GEO-CM04S1 versus BNT162b2. Ongoing larger studies must confirm the generalizability of these promising results. GEO-CM04S1's property to elicit enhanced cellular immunity may be key to protecting CLL individuals against severe COVID-19 and SARS-CoV-2 escape mutants. Sandra Ortega-Francisco, Maria Teresa Prudente de Aquino, Corinna La Rosa, Don J. Diamond authored the manuscript; Flavia Chiuppesi, Sandra Thomas, Alexey Danilov authored the clinical trial protocol; Miguel-Angel Gutierrez, Jacqueline Miller, Jing Li, Jada Mack-Onyeike, Yifei Zhou and Qiao Zhou conducted experiments; Alexey Danilov, Stephen Rosen and Tanya Siddiqi enrolled patients; Dongyun Yang and Wasima Rida conducted statistical analysis; Corinna La Rosa supervised sample processing; Shannon Dempsey submitted regulatory documents; Teodora Kaltcheva synthesized peptides; Kelly McKee and Erica Raiden were the liaisons for the GeoVax vaccine sponsor as well as responsible for overseeing trial protocol, immune testing and results; Angela Patterson directed procurement and financial liaison with correlatives funder; and Felix Wussow was involved with vaccine assessment. The authors thank the Briskin Foundation for supporting clinical activities. The authors thank the patients for their contribution by participating in the trial. Nurses, physicians and other clinical professionals are acknowledged for their dedication to the patients and the clinical trial integrity. The authors acknowledge and thank the members of the COH clinical manufacturing facility for dedicated effort in producing the vaccine for patient use. The authors thank Alba Grifoni and Alessandro Sette for providing SARS-CoV-2 Spike (S) megapool peptides used in flow cytometry experiments. Don J. Diamond, Felix Wussow and Flavia Chiuppesi have patents that are licensed to Geovax. Don J. Diamond receives research support and consulting fees from Geovax. Sandra Ortega-Francisco and Wasima Rida receive consulting fees from Geovax. Alexey Danilov has received consulting fees from Abbvie, ADCT, AstraZeneca, BeiGene, Bristol-Meyers-Squibb, GenMab, Incyte, Janssen, Lilly Oncology, MEI Pharma, Merck, Nurix, Regeneron and Roche and has ongoing research funding from Briskin Foundation, Abbvie, AstraZeneca, BeiGene, GenMab, Incyte, Lilly Oncology, Merck, Nurix and Regeneron. Erica Raiden and Kelly McKee are employees of Geovax. Flavia Chiuppesi is currently an employee of GSK Inc. All the other authors have no conflicts of interest to disclose. The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions. Data S1. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.