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•A single Ad26.COV2.S booster given 45–75 or 90–240 days after 2 BBIBP-CorV doses was well tolerated.•Robust humoral and cell-mediated immune responses were measured at day 28 in both interval groups.•Humoral responses were strongest against ancestral virus, followed by delta then omicron variants.•T-cell–produced IFN-γ increased ≈10-fold in both groups after this heterologous booster dose. 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. 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. 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. 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.

•An Ad26.COV2.S booster was given 45–75 or 90–240 days after 2 BBIBP-CorV doses.•Humoral booster responses trended higher after the longer pre-boost interval and against ancestral virus.•Durable T-cell responses were detected in both interval groups.•Hybrid immune responses were higher than booster-effect responses.•Immune responses trended lower but were durable in adults ≥ 60 years. 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. 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). 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. 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.

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.

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.

The randomized trial assessing the efficacy of a single injection of the Ad26.COV2.S showed 56.3% vaccine efficacy beginning 14 days after injection and 52.9% efficacy more than 28 days after injection against moderate to severe–critical Covid-19. Protection lasted at least 6 months without an added boost. Vaccination was associated with mild-to-moderate adverse effects.

Ad26.COV2.S vaccine is a replication-incompetent human adenovirus type 26 vector containing the gene sequence that produces SARS-CoV-2 spike protein in a prefusion-stabilized conformation. In a randomized trial involving nearly 40,000 persons, vaccine efficacy was 66% against moderate to severe–critical Covid-19 and 85% against severe–critical Covid-19. Efficacy against the variant first identified in South Africa was 64% against moderate disease and 82% against severe–critical disease.

DNA-Vector transfection of eukaryotic cells needs transport of the DNA to the nucleus for efficient transcription and subsequent translation. In suspension cells most DNA-transfection methods are either inefficient (e.g. lipofection) or stressful for the cells (electroporation or nucleofection), which leads to high cell losses during the transfection process. We report the use of in vitro transcribed, capped and polyadenylated mRNA for the transfection of leukemic cell lines in comparison to DNA delivery. Templates for in vitro transcription were generated by PCR. The forward primer included the T7-promoter and the reverse primer a fragment of the beta-globin 3'UTR to increase the in vivo half life of the mRNA. Electroporation of GFP-mRNA into Jurkat, K562 or Kg1a lead to a GFP overexpression in 95 % of the transfected cells, with survival rates of 80 to 90%. Electroporation with pEGFP-N1 using the same pulse conditions resulted in GFP-overexpression in 62% of Jurkat or 9% of K562, with survival rates of 50% and 40 %, respectively. Our results suggest that electroporation of DNA is more toxic than mRNA transfection. GFP-expression in mRNA transfected cells was visible two hours after the transfection and peaked at about 12h. In contrast to DNA-transfection, mRNA mediated GFP expression levels were uniform in transfected cells. To investigate whether mRNA transfection results in functional transgene expression, mRNA coding the chemokine receptor CXCR4 was generated and electroporated into CXCR4 negative K562 cells. Flow cytometric analyses showed CXCR4 overexpression in more than 90% of transfected cells. Calcium flux measurements displayed a robust increase of intracellular free calcium in CXCR4-mRNA transfected K562 upon binding of the CXCR4 ligand SDF-1, while no calcium flux was visible in GFP-mRNA transfected or untransfected K562. Electroporation of GFP-mRNA into CD34+ hematopoietic progenitor cells or mesenchymal stem cells resulted in GFP overexpression in > 50% of transfected cells, respectively. Experiments are under way to further improve the mRNA transfection for primary cells. mRNA transfection is a highly efficient means for transient overexpression of transgenes in various cell lines and primary 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.

Wild-type murine onco-retroviruses cause cancer in part by insertional mutagenesis, but until recently, cancers had not been observed in humans or animals subjected to marking or treatment with the highly modified murine retrovirus vectors in current use. Recently, mutagenesis activation of the LMO-2 gene was observed to be associated with the development of lymphoid leukemia several years after two infants with X-SCID had their immune systems restored by gene therapy with a therapeutic onco-retrovirus vector. Insertional mutagenesis activation of other oncogenes associated with development of cancers has been reported in vector-marked mice. These observations highlight the importance of encouraging many investigators to conduct large animal marking studies which follow the insertional pattern of vectors planned for human treatments. We have recently reported that a very high titer RD114 pseudotyped MFGS-gp91phox vector achieves unprecedented levels of correction of the oxidase defect in CD34+ cells from patients with X-linked chronic granulomatous disease in the NOD/SCID mouse model. In anticipation of considering clinical use of this vector, we transduced mobilized peripheral blood CD34+ cells (PBSC CD34+) of two healthy rhesus macaques with the RD114-MFGS-gp91phox. Transduced PBSC CD34+ with an average two vector copies per cell were transplanted on culture day 4 into two non-myeloablative (2× 300 Rads) irradiated animals. The moderately high levels of in vivo marking (>6%) seen in the first months following gene marking decreased and stabilized at about 8 months post transplant. Marking for animal J976 and D406 at 15 and 19 months, respectively is 1.3% and 0.3% in B lymphocytes, 0.3% and 0.3% in neutrophils and 0.7% and 0.4% in T lymphocytes at which time the animals remained healthy. Vector insertion analysis was performed by LAM-PCR, using gel electrophoresis and GeneScan analysis to demonstrate that despite the relative low marking at 10 months and later animal D406 had more than 25 and animal J976 had more than 40 separate insertional events. 34% of integration sites are located in coding regions. No predominant clone was seen to emerge over time and no patterns of insertion site preferences were detected up to this point.

Penetration of surface meltwater to the bed of the Greenland Ice Sheet each summer causes an initial increase in ice speed due to elevated basal water pressure, followed by slowdown in late summer that continues into fall and winter. While this seasonal pattern is commonly explained by an evolution of the subglacial drainage system from an inefficient distributed to efficient channelized configuration, mounting evidence indicates that subglacial channels are unable to explain important aspects of hydrodynamic coupling in late summer and fall. Here we use numerical models of subglacial drainage and ice flow to show that limited, gradual leakage of water and lowering of water pressure in weakly connected regions of the bed can explain the dominant features in late and post melt season ice dynamics. These results suggest that a third weakly connected drainage component should be included in the conceptual model of subglacial hydrology.