Heterologous Ad26.COV2.S booster after primary BBIBP-CorV vaccination against SARS-CoV-2 infection: 1-year follow-up of a phase 1/2 open-label trial.

BACKGROUND: Inactivated whole-virus vaccination elicits immune responses to both SARS-CoV-2 nucleocapsid (N) and spike (S) proteins, like natural infections. A heterologous Ad26.COV2.S booster given at two different intervals after primary BBIBP-CorV vaccination was safe and immunogenic at days 28 and 84, with higher immune responses observed after the longer pre-boost interval. We describe booster-specific and hybrid immune responses over 1 year. METHODS: This open-label phase 1/2 study was conducted in healthy Thai adults aged ≥ 18 years who had completed primary BBIBP-CorV primary vaccination between 90-240 (Arm A1; n = 361) or 45-75 days (Arm A2; n = 104) before enrolment. All received an Ad26.COV2.S booster. We measured anti-S and anti-N IgG antibodies by Elecsys®, neutralizing antibodies by SARS-CoV-2 pseudovirus neutralization assay, and T-cell responses by quantitative interferon (IFN)-γ release assay. Immune responses were evaluated in the baseline-seronegative population (pre-booster anti-N < 1.4 U/mL; n = 241) that included the booster-effect subgroup (anti-N < 1.4 U/mL at each visit) and the hybrid-immunity subgroup (anti-N ≥ 1.4 U/mL and/or SARS-CoV-2 infection, irrespective of receiving non-study COVID-19 boosters). RESULTS: In Arm A1 of the booster-effect subgroup, anti-S GMCs were 131-fold higher than baseline at day 336; neutralizing responses against ancestral SARS-CoV-2 were 5-fold higher than baseline at day 168; 4-fold against Omicron BA.2 at day 84. IFN-γ remained approximately 4-fold higher than baseline at days 168 and 336 in 18-59-year-olds. Booster-specific responses trended lower in Arm A2. In the hybrid-immunity subgroup at day 336, anti-S GMCs in A1 were 517-fold higher than baseline; neutralizing responses against ancestral SARS-CoV-2 and Omicron BA.2 were 28- and 31-fold higher, respectively, and IFN-γ was approximately 14-fold higher in 18-59-year-olds at day 336. Durable immune responses trended lower in ≥ 60-year-olds. CONCLUSION: A heterologous Ad26.COV2.S booster after primary BBIBP-CorV vaccination induced booster-specific immune responses detectable up to 1 year that were higher in participants with hybrid immunity. CLINICAL TRIALS REGISTRATION: NCT05109559.

Single Ad26.COV2.S booster dose following two doses of BBIBP-CorV vaccine against SARS-CoV-2 infection in adults: Day 28 results of a phase 1/2 open-label trial.

BACKGROUND: The inactivated COVID-19 whole-virus vaccine BBIBP-CorV has been extensively used worldwide. Heterologous boosting after primary vaccination can induce higher immune responses against SARS-CoV-2 than homologous boosting. The safety and immunogenicity after 28 days of a single Ad26.COV2.S booster dose given at different intervals after 2 doses of BBIBP-CorV are presented. METHODS: This open-label phase 1/2 trial was conducted in healthy adults in Thailand who had completed 2-dose primary vaccination with BBIBP-CorV. Participants received a single booster dose of Ad26.COV2.S (5 × 1010 virus particles) 90-240 days (Group A1; n = 360) or 45-75 days (Group A2; n = 66) after the second BBIBP-CorV dose. Safety and immunogenicity were assessed over 28 days. Binding IgG antibodies to the full-length pre-fusion Spike and anti-nucleocapsid proteins of SARS-CoV-2 were measured by enzyme-linked immunosorbent assay. The SARS-CoV-2 pseudovirus neutralization assay and live virus microneutralization assay were used to quantify the neutralizing activity of antibodies against ancestral SARS-CoV-2 (Wuhan-Hu-1) and the delta (B.1.617.2) and omicron (B.1.1.529/BA.1 and BA.2) variants. The cell-mediated immune response was measured using a quantitative interferon (IFN)-γ release assay in whole blood. RESULTS: Solicited local and systemic adverse events (AEs) on days 0-7 were mostly mild, as were unsolicited vaccine-related AEs during days 0-28, with no serious AEs. On day 28, anti-Spike binding antibodies increased from baseline by 487- and 146-fold in Groups A1 and A2, and neutralizing antibodies against ancestral SARS-CoV-2 by 55- and 37-fold, respectively. Humoral responses were strongest against ancestral SARS-CoV-2, followed by the delta, then the omicron BA.2 and BA.1 variants. T-cell-produced interferon-γ increased approximately 10-fold in both groups. CONCLUSIONS: A single heterologous Ad26.COV2.S booster dose after two BBIBP-CorV doses was well tolerated and induced robust humoral and cell-mediated immune responses measured at day 28 in both interval groups. CLINICAL TRIALS REGISTRATION: NCT05109559.

Final Analysis of Efficacy and Safety of Single-Dose Ad26.COV2.S.

BACKGROUND: The Ad26.COV2.S vaccine was highly effective against severe-critical coronavirus disease 2019 (Covid-19), hospitalization, and death in the primary phase 3 efficacy analysis. METHODS: We conducted the final analysis in the double-blind phase of our multinational, randomized, placebo-controlled trial, in which adults were assigned in a 1:1 ratio to receive single-dose Ad26.COV2.S (5×1010 viral particles) or placebo. The primary end points were vaccine efficacy against moderate to severe-critical Covid-19 with onset at least 14 days after administration and at least 28 days after administration in the per-protocol population. Safety and key secondary and exploratory end points were also assessed. RESULTS: Median follow-up in this analysis was 4 months; 8940 participants had at least 6 months of follow-up. In the per-protocol population (39,185 participants), vaccine efficacy against moderate to severe-critical Covid-19 at least 14 days after administration was 56.3% (95% confidence interval [CI], 51.3 to 60.8; 484 cases in the vaccine group vs. 1067 in the placebo group); at least 28 days after administration, vaccine efficacy was 52.9% (95% CI, 47.1 to 58.1; 433 cases in the vaccine group vs. 883 in the placebo group). Efficacy in the United States, primarily against the reference strain (B.1.D614G) and the B.1.1.7 (alpha) variant, was 69.7% (95% CI, 60.7 to 76.9); efficacy was reduced elsewhere against the P.1 (gamma), C.37 (lambda), and B.1.621 (mu) variants. Efficacy was 74.6% (95% CI, 64.7 to 82.1) against severe-critical Covid-19 (with only 4 severe-critical cases caused by the B.1.617.2 [delta] variant), 75.6% (95% CI, 54.3 to 88.0) against Covid-19 leading to medical intervention (including hospitalization), and 82.8% (95% CI, 40.5 to 96.8) against Covid-19-related death, with protection lasting 6 months or longer. Efficacy against any severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection was 41.7% (95% CI, 36.3 to 46.7). Ad26.COV2.S was associated with mainly mild-to-moderate adverse events, and no new safety concerns were identified. CONCLUSIONS: A single dose of Ad26.COV2.S provided 52.9% protection against moderate to severe-critical Covid-19. Protection varied according to variant; higher protection was observed against severe Covid-19, medical intervention, and death than against other end points and lasted for 6 months or longer. (Funded by Janssen Research and Development and others; ENSEMBLE ClinicalTrials.gov number, NCT04505722.).

Safety and Efficacy of Single-Dose Ad26.COV2.S Vaccine against Covid-19.

BACKGROUND: The Ad26.COV2.S vaccine is a recombinant, replication-incompetent human adenovirus type 26 vector encoding full-length severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein in a prefusion-stabilized conformation. METHODS: In an international, randomized, double-blind, placebo-controlled, phase 3 trial, we randomly assigned adult participants in a 1:1 ratio to receive a single dose of Ad26.COV2.S (5×1010 viral particles) or placebo. The primary end points were vaccine efficacy against moderate to severe-critical coronavirus disease 2019 (Covid-19) with an onset at least 14 days and at least 28 days after administration among participants in the per-protocol population who had tested negative for SARS-CoV-2. Safety was also assessed. RESULTS: The per-protocol population included 19,630 SARS-CoV-2-negative participants who received Ad26.COV2.S and 19,691 who received placebo. Ad26.COV2.S protected against moderate to severe-critical Covid-19 with onset at least 14 days after administration (116 cases in the vaccine group vs. 348 in the placebo group; efficacy, 66.9%; adjusted 95% confidence interval [CI], 59.0 to 73.4) and at least 28 days after administration (66 vs. 193 cases; efficacy, 66.1%; adjusted 95% CI, 55.0 to 74.8). Vaccine efficacy was higher against severe-critical Covid-19 (76.7% [adjusted 95% CI, 54.6 to 89.1] for onset at ≥14 days and 85.4% [adjusted 95% CI, 54.2 to 96.9] for onset at ≥28 days). Despite 86 of 91 cases (94.5%) in South Africa with sequenced virus having the 20H/501Y.V2 variant, vaccine efficacy was 52.0% and 64.0% against moderate to severe-critical Covid-19 with onset at least 14 days and at least 28 days after administration, respectively, and efficacy against severe-critical Covid-19 was 73.1% and 81.7%, respectively. Reactogenicity was higher with Ad26.COV2.S than with placebo but was generally mild to moderate and transient. The incidence of serious adverse events was balanced between the two groups. Three deaths occurred in the vaccine group (none were Covid-19-related), and 16 in the placebo group (5 were Covid-19-related). CONCLUSIONS: A single dose of Ad26.COV2.S protected against symptomatic Covid-19 and asymptomatic SARS-CoV-2 infection and was effective against severe-critical disease, including hospitalization and death. Safety appeared to be similar to that in other phase 3 trials of Covid-19 vaccines. (Funded by Janssen Research and Development and others; ENSEMBLE ClinicalTrials.gov number, NCT04505722.).

Serum albumin strongly influences SDF-1 dependent migration.

Stem cell migration is largely regulated by the chemokine SDF-1 and its receptor CXCR4. In the present study, we analyzed the effect of protein on SDF-1 dependent chemotaxis using CXCR4 expressing primary CD34+ hematopoietic progenitor cells for transwell migration assays. We show that migration towards SDF-1 is abolished in the absence of protein, while addition of serum albumin rescues SDF-1 dependent migration. Acid hydrolyzation or tryptic digest of protein eliminates its migration supporting effect, showing that the intact protein is necessary. We demonstrate that gradients of human serum albumin (HSA) that are physiologically present in vivo between human plasma and interstitial fluid (bone marrow) greatly influence SDF-1 dependent migration of hematopoietic progenitor cells. While SDF-1 dependent migration is strongly enhanced in the presence of a HSA gradient from 4% (plasma) towards 1% (interstitial fluid), reversion of the protein concentrations inhibits SDF-1 dependent chemotaxis. Furthermore, migration is induced to lower serum albumin concentrations in the presence of equal SDF-1 concentration, while albumin gradients in the absence of SDF-1 have no effect. Our results suggest that physiological gradients of serum albumin between blood and bone marrow support SDF-1 dependent homing of hematopoietic progenitor cells to the stem cell niche.

S1P(1) overexpression stimulates S1P-dependent chemotaxis of human CD34+ hematopoietic progenitor cells but strongly inhibits SDF-1/CXCR4-dependent migration and in vivo homing.

The CXC chemokine receptor 4 (CXCR4) and its ligand stromal derived factor 1 (SDF-1) regulate egress and homing of hematopoietic stem cells. Activation of sphingosine-1-phosphate (S1P) receptors (S1P(1-5)) modulates chemokine-induced migration of lymphocytes and hematopoietic stem cells. To analyze the influence of S1P(1) on SDF-1-dependent chemotaxis and trafficking, we overexpressed S1P(1) in CD34+ mobilized peripheral blood progenitor cells (PBPCs). Using a gamma-retroviral vector, transgene overexpression was achieved in more than 90% of target cells. S1P(1) transgene positive PBPCs showed enhanced chemotaxis towards S1P. S1P(1) overexpression resulted in reduced CXCR4 surface expression levels and strong inhibition of SDF-1-dependent ERK1/2 phosphorylation and Ca(2+) flux. Furthermore, SDF-1-dependent migration of S1P(1) overexpressing PBPCs or Jurkat cells was reduced up to 10-fold. Sublethally irradiated NOD/SCID mice were transplanted with 6-day cultured PBPCs overexpressing either S1P(1)-IRES-GFP or GFP alone. Screening for GFP positive human cells in the mouse bone marrow 20h after transplantation revealed an eightfold reduction in bone marrow homing of S1P(1) transgene expressing cells. Our data suggest that S1P(1) acts as an inhibitor of CXCR4-dependent migration of hematopoietic cells to sites of SDF-1 production.

mRNA transfection of CXCR4-GFP fusion--simply generated by PCR-results in efficient migration of primary human mesenchymal stem cells.

We present a general, entirely PCR-based strategy to construct mRNAs coding for green fluorescent protein (GFP) fusion proteins from a cDNA pool. We exemplify our approach for the chemokine receptor CXCR4. mRNA transfection of the PCR-generated fusion of CXCR4-GFP into K562 cells or primary mesenchymal stem cells (MSCs) resulted in excellent viability (> 90%) with more than 90% of target cells expressing easily detectable CXCR4-GFP for > 72 h. The fusion protein was localized in the plasma membrane and was rapidly internalized upon incubation with the CXCR4 ligand stromal cell-derived factor-1 (SDF-1). Transwell migration experiments showed significantly increased migration of CXCR4-GFP mRNA-transfected MSCs toward a gradient of SDF-1, demonstrating that mRNA-mediated chemokine receptor overexpression allows for transient initiation of chemotaxis. The presented strategy to construct a PCR-based fluorescent fusion protein can be generally applied to other genes of interest to study their function by simple overexpression and easy detection in primary cells.

Gene therapy for chronic granulomatous disease.

Patients with chronic granulomatous disease (CGD) cannot generate reactive oxygen metabolites, and suffer from severe recurrent infections and dysregulated inflammation. Haematopoietic stem cell transplantation is the only established option for definitive cure for patients with a suitable donor and is indicated when conventional prophylaxis and therapy with antimicrobial medication fail. Gene therapy has the potential to cure CGD, and several clinical trials have been conducted since 1997. Whereas initial studies resulted in low and short-term engraftment of CGD-corrected cells, recent trials demonstrated clinical benefit when engraftment was enhanced by busulfan conditioning prior to infusion of gene-corrected cells. However, the progress in gene therapy has been hampered by the appearance of insertional mutagenesis causing leukaemia in a trial for patients with X-linked severe combined immunodeficiency and by the emergence of dominant clones in a recent trial for the X-linked form of CGD. These findings stimulated the development of modified vector systems that demonstrate reduced genotoxicity in vitro and in animal models. New gene therapy protocols that allow efficient gene transfer and durable expression but limit the risk for insertional mutagenesis are envisioned to become an important therapeutic option for patients with CGD.

The late dividing population of gamma-retroviral vector transduced human mobilized peripheral blood progenitor cells contributes most to gene-marked cell engraftment in nonobese diabetic/severe combined immunodeficient mice.

We used the nonobese diabetic/severe combined immunodeficient (NOD/SCID) mouse model to assess the repopulation potential of subpopulations of mobilized human CD34+ peripheral blood progenitor cells (PBPC). First, PBPC were transduced with gamma-retrovirus vector RD114-MFGS-CFP, which requires cell division for successful transduction, at 24 hours, 48 hours, and 72 hours to achieve 96% cyan fluorescent protein (CFP)-positive cells. Cells were sorted 12 hours after the last transduction into CFP-positive (divided cells) and CFP-negative populations. CFP-positive cells were transplanted postsort, whereas the CFP-negative cells were retransduced and injected at 120 hours. The CFP-negative sorted and retransduced cells contained markedly fewer vector copies and resulted in a 32-fold higher overall engraftment and in a 13-fold higher number of engrafted transgene positive cells. To assess cell proliferation as an underlying cause for the different engraftment levels, carboxyfluorescein succinimidyl ester-labeling of untransduced PBPC was performed to track the number of cell divisions. At 72 hours after initiation of culture, when 95% of all cells have divided, PBPC were sorted into nondivided and divided fractions and transplanted into NOD/SCID mice. Nondivided cells demonstrated 45-fold higher engraftment than divided cells. Late dividing PBPC in ex vivo culture retain high expression of the stem cell marker CD133, whereas rapidly proliferating cells lose CD133 in correlation to the number of cell divisions. Our studies demonstrate that late dividing progenitors transduced with gamma-retroviral vectors contribute most to NOD/SCID engraftment and transgene marking. Confining the gamma-retroviral transduction to CD133-positive cells on days 3 and 4 could greatly reduce the number of transplanted vector copies, limiting the risk of leukemia from insertional mutagenesis. Disclosure of potential conflicts of interest is found at the end of this article.

Polyclonal long-term MFGS-gp91phox marking in rhesus macaques after nonmyeloablative transplantation with transduced autologous peripheral blood progenitor cells.

We have recently reported that the RD114-pseudotyped MFGS-gp91phox vector achieves unprecedented levels of correction of the NADPH-oxidase gp91phox (approved gene symbol CYBB) defect in CD34(+) cells from patients with X-linked chronic granulomatous disease in the NOD/SCID mouse model. Considering clinical use of this vector, we transplanted autologous mobilized peripheral blood CD34(+) progenitor cells, transduced with the RD114-MFGS-gp91phox vector, into two healthy rhesus macaques following nonmyeloablative conditioning. The moderately high levels of in vivo marking seen in the first months following transduction decreased and stabilized at about 8 months posttransplant. Marking for both healthy animals after 15 months was 0.3 to 1.3 vector copies per 100 cells in lymphocytes, neutrophils, and monocytes. Vector insertion analyses performed by linear amplification-mediated PCR and sequencing identified 32 and 45 separate insertion sites in the animals. Identical insertion sites were found in myeloid cells and lymphocytes, demonstrating the successful transduction of lymphomyeloid progenitors. Some inserts landed in the vicinity of genes controlling cell cycle and proliferation. Statistical analyses of insertion sites 1 year posttransplant suggest a high diversity of insertion sites despite low marking.

CXCR4-transgene expression significantly improves marrow engraftment of cultured hematopoietic stem cells.

Hematopoietic stem cells (HSCs) lose marrow reconstitution potential during ex vivo culture. HSC migration to stromal cell-derived factor (SDF)-1 (CXCL12) correlates with CXC chemokine receptor 4 (CXCR4) expression and marrow engraftment. We demonstrate that mobilized human CD34+ peripheral blood stem cells (CD34+ PBSCs) lose CXCR4 expression during prolonged culture. We transduced CD34+ PBSCs with retrovirus vector encoding human CXCR4 and achieved 18-fold more CXCR4 expression in over 87% of CD34+ cells. CXCR4-transduced cells yielded increased calcium flux and up to a 10-fold increase in migration to SDF-1. Six-day cultured CXCR4-transduced cells demonstrated significant engraftment in nonobese diabetic/severe combined immunodeficient mice under conditions in which control transduced cells resulted in low or no engraftment. We conclude that transduction-mediated overexpression of CXCR4 significantly improves marrow engraftment of cultured PBSCs.