Safety and immune response kinetics of GRAd-COV2 vaccine: phase 1 clinical trial results.

Despite the successful deployment of efficacious vaccines and therapeutics, the development of novel vaccines for SARS-CoV-2 remains a major goal to increase vaccine doses availability and accessibility for lower income setting. We report here on the kinetics of Spike-specific humoral and T-cell response in young and old volunteers over 6 months follow-up after a single intramuscular administration of GRAd-COV2, a gorilla adenoviral vector-based vaccine candidate currently in phase-2 of clinical development. At all three tested vaccine dosages, Spike binding and neutralizing antibodies were induced and substantially maintained up to 3 months, to then contract at 6 months. Potent T-cell responses were readily induced and sustained throughout the study period, with only minor decline. No major differences in immune response to GRAd-COV2 vaccination were observed in the two age cohorts. In light of its favorable safety and immunogenicity, GRAd-COV2 is a valuable candidate for further clinical development and potential addition to the COVID-19 vaccine toolbox to help fighting SARS-CoV-2 pandemic.

Engineered Sleeping Beauty Transposon as Efficient System to Optimize Chimp Adenoviral Production.

Sleeping Beauty (SB) is the first DNA transposon employed for efficient transposition in vertebrate cells, opening new applications for genetic engineering and gene therapies. A transposon-based gene delivery system holds the favourable features of non-viral vectors and an attractive safety profile. Here, we employed SB to engineer HEK293 cells for optimizing the production of a chimpanzee Adenovector (chAd) belonging to the Human Mastadenovirus C species. To date, chAd vectors are employed in several clinical settings for infectious diseases, last but not least COVID-19. A robust, efficient and quick viral vector production could advance the clinical application of chAd vectors. To this aim, we firstly swapped the hAd5 E1 with chAd-C E1 gene by using the CRISPR/Cas9 system. We demonstrated that in the absence of human Ad5 E1, chimp Ad-C E1 gene did not support HEK293 survival. To improve chAd-C vector production, we engineered HEK293 cells to stably express the chAd-C precursor terminal protein (ch.pTP), which plays a crucial role in chimpanzee Adenoviral DNA replication. The results indicate that exogenous ch.pTP expression significantly ameliorate the packaging and amplification of recombinant chAd-C vectors thus, the engineered HEK293ch.pTP cells could represent a superior packaging cell line for the production of these vectors.

GRAd-COV2, a gorilla adenovirus-based candidate vaccine against COVID-19, is safe and immunogenic in younger and older adults.

Safe and effective vaccines against coronavirus disease 2019 (COVID-19) are essential for ending the ongoing pandemic. Although impressive progress has been made with several COVID-19 vaccines already approved, it is clear that those developed so far cannot meet the global vaccine demand alone. We describe a COVID-19 vaccine based on a replication-defective gorilla adenovirus expressing the stabilized prefusion severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein named GRAd-COV2. We assessed the safety and immunogenicity of a single-dose regimen of this vaccine in healthy younger and older adults to select the appropriate dose for each age group. For this purpose, a phase 1, dose-escalation, open-labeled trial was conducted including 90 healthy participants (45 aged 18 to 55 years old and 45 aged 65 to 85 years old) who received a single intramuscular administration of GRAd-COV2 at three escalating doses. Local and systemic adverse reactions were mostly mild or moderate and of short duration, and no serious adverse events were reported. Four weeks after vaccination, seroconversion to spike protein and receptor binding domain was achieved in 43 of 44 young volunteers and in 45 of 45 older participants. Consistently, neutralizing antibodies were detected in 42 of 44 younger-age and 45 of 45 older-age volunteers. In addition, GRAd-COV2 induced a robust and T helper 1 cell (TH1)­skewed T cell response against the spike protein in 89 of 90 participants from both age groups. Overall, the safety and immunogenicity data from the phase 1 trial support the further development of this vaccine.

Immunogenicity of a new gorilla adenovirus vaccine candidate for COVID-19.

The coronavirus disease 2019 (COVID-19) pandemic caused by the emergent severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) threatens global public health, and there is an urgent need to develop safe and effective vaccines. Here, we report the generation and the preclinical evaluation of a novel replication-defective gorilla adenovirus-vectored vaccine encoding the pre-fusion stabilized Spike (S) protein of SARS-CoV-2. We show that our vaccine candidate, GRAd-COV2, is highly immunogenic both in mice and macaques, eliciting both functional antibodies that neutralize SARS-CoV-2 infection and block Spike protein binding to the ACE2 receptor, and a robust, T helper (Th)1-dominated cellular response. We show here that the pre-fusion stabilized Spike antigen is superior to the wild type in inducing ACE2-interfering, SARS-CoV-2-neutralizing antibodies. To face the unprecedented need for vaccine manufacturing at a massive scale, different GRAd genome deletions were compared to select the vector backbone showing the highest productivity in stirred tank bioreactors. This preliminary dataset identified GRAd-COV2 as a potential COVID-19 vaccine candidate, supporting the translation of the GRAd-COV2 vaccine in a currently ongoing phase I clinical trial (ClinicalTrials.gov: NCT04528641).

Characterization of pUL5, an HCMV protein interacting with the cellular protein IQGAP1.

Among the Herpesviridae, human cytomegalovirus (HCMV) owns the largest genome and displays a huge coding potential. Here, we characterized the UL5 gene product (pUL5) of the clinical isolate TR strain. The protein was predicted as a 166-amino-acid membrane protein with a theoretical mass of 19 kDa. Recombinant virus expressing pUL5 with a tag allowed the identification of two pUL5 non-glycosylated species of approximately 19 and 9 kDa, expressed with early and late kinetic respectively. Experiments in infection confirmed that the lower molecular weight species was translated from an internal ATG in the UL5 open reading frame. Confocal microscopy analysis showed that pUL5 localized within the assembly compartment, but is not incorporated in the virion, as shown by Western blot on purified viral particles. Finally, pull-down experiments coupled with mass spectrometry analysis identified IQGAP1 as a pUL5 interactor, giving new hints on possible roles of pUL5 during HCMV infection.

Testosterone Plasma Concentration is Associated with Insulin Resistance in Male Hypertensive Patients.

Background: Low testosterone levels are a common finding among men with Type 2 Diabetes Mellitus (T2DM) and are inversely related to insulin resistance. Whether this relationship holds true in patients with hypertension, but normal glucose tolerance or prediabetes, is unclear. Methods: We recruited 87 male outpatients with essential arterial hypertension, aged 35-70 years. Anthropometric data were collected, an Oral Glucose Tolerance Test (OGTT) performed, and the homeostasis model assessment of insulin resistance (HOMA-IR) score calculated. Follicle-Stimulating Hormone, Luteinizing Hormone, testosterone, Sex Hormone-Binding-Globulin and free-testosterone were measured. The concentrations of sex hormones were compared between normoglucotolerant, prediabetic and diabetic patients. Non-parametric tests were applied as appropriate to verify differences among groups, while multiple linear regression was used to predict the variability of testosterone and free-testosterone. Results: Total serum testosterone concentration was significantly lower in T2DM in comparison to normoglucotolerant subjects (p<0.01) and was inversely related to body mass index (r=- 0.25, p<0.01), waist circumference (r=- 0.27, p<0.01), pre and post-OGTT plasma glucose (r=- 0.4, p<0.0001 and r=- 0.29, p<0.01, respectively), pre and post-OGTT plasma insulin (r=- 0.42, p<0.0001 and r=- 0.42, p<0.0001) and HOMA-IR (r=- 0.46, p<0.0001). Similar associations were observed for free testosterone; HOMA-IR was related to testosterone and free-testosterone even in patients with normal glucose tolerance (r=- 0.47, p<0.01 and r=- 0.34, p<0.05, respectively). At multivariate analysis HOMA-IR was the only variable associated to testosterone (p<0.001) and free-testosterone (p<0.05) plasma concentration. Conclusions: In males with hypertension, the link between insulin sensitivity and hypothalamic-pituitary-gonadal axis is maintained along the entire spectrum of glucose tolerance.

Human cytomegalovirus pUL10 interacts with leukocytes and impairs TCR-mediated T-cell activation.

Human cytomegalovirus (HCMV) is known to exert suppressive effects on the host immune system through expression of various viral genes, thus directly and indirectly affecting antiviral immunity of the infected individuals. We report here that HCMV UL10 encodes a protein (pUL10) with immunosuppressive properties. UL10 has been classified as a member of the HCMV RL11 gene family. Although pUL10 is known to be dispensable for viral replication in cultured cells, its amino-acid sequence is well conserved among different HCMV isolates, suggesting that the protein has a crucial role in viral survival in the host environment. We show that pUL10 is cleaved from the cell surface of fibroblasts as well as epithelial cells and interacts with a cellular receptor ubiquitously expressed on the surface of human leukocytes, demonstrated by ex vivo cell-based assays and flow cytometric analyses on both lymphoid cell lines and primary blood cells. Furthermore, preincubation of peripheral blood mononuclear cells with purified pUL10 ectodomain results in significantly impaired proliferation and substantially reduced pro-inflammatory cytokine production, in particular in CD4+ T cells upon in vitro T-cell stimulation. The inhibitory effect of pUL10 is also observed on antigen receptor-mediated intracellular tyrosine phosphorylation in a T-cell line. Based on these observations, we suggest that pUL10 is a newly identified immunomodulatory protein encoded by HCMV. Further elucidation of interactions between pUL10 and the host immune system during HCMV may contribute to finding ways towards new therapies for HCMV infection.

The Human Cytomegalovirus UL116 Gene Encodes an Envelope Glycoprotein Forming a Complex with gH Independently from gL.

UNLABELLED: Human cytomegalovirus (HCMV) is a major cause of morbidity and mortality in transplant patients and is the leading viral cause of birth defects after congenital infection. HCMV infection relies on the recognition of cell-specific receptors by one of the viral envelope glycoprotein complexes. Either the gH/gL/gO or the gH/gL/UL128/UL130/UL131A (Pentamer) complex has been found to fulfill this role, accounting for HCMV entry into almost all cell types. We have studied the UL116 gene product, a putative open reading frame identified by in silico analysis and predicted to code for a secreted protein. Virus infection experiments in mammalian cells demonstrated that UL116 is expressed late in the HCMV replication cycle and is a heavily glycosylated protein that first localizes to the cellular site of virus assembly and then inserts into the virion envelope. Transient-transfection studies revealed that UL116 is efficiently transported to the plasma membrane when coexpressed with gH and that gL competes with UL116 for gH binding. Further evidence for gH/UL116 complex formation was obtained by coimmunoprecipitation experiments on both transfected and infected cells and biochemical characterization of the purified complex. In summary, our results show that the product of the UL116 gene is an HCMV envelope glycoprotein that forms a novel gH-based complex alternative to gH/gL. Remarkably, the gH/UL116 complex is the first herpesvirus gH-based gL-less complex. IMPORTANCE: HCMV infection can cause severe disease in immunocompromised adults and infants infected in utero The dissection of the HCMV entry machinery is important to understand the mechanism of viral infection and to identify new vaccine antigens. The gH/gL/gO and gH/gL/UL128/UL130/UL131 (Pentamer) complexes play a key role in HCMV cell entry and tropism. Both complexes are formed by an invariant gH/gL scaffold on which the other subunits assemble. Here, we show that the UL116 gene product is expressed in infected cells and forms a heterodimer with gH. The gH/UL116 complex is carried on the infectious virions, although in smaller amounts than gH/gL complexes. No gH/UL116/gL ternary complex formed in transfected cells, suggesting that the gH/UL116 complex is independent from gL. This new gH-based gL-free complex represents a potential target for a protective HCMV vaccine and opens new perspectives on the comprehension of the HCMV cell entry mechanism and tropism.