Safety and immunogenicity of the protein-based PHH-1V compared to BNT162b2 as a heterologous SARS-CoV-2 booster vaccine in adults vaccinated against COVID-19: a multicentre, randomised, double-blind, non-inferiority phase IIb trial.

Background: A SARS-CoV-2 protein-based heterodimer vaccine, PHH-1V, has been shown to be safe and well-tolerated in healthy young adults in a first-in-human, Phase I/IIa study dose-escalation trial. Here, we report the interim results of the Phase IIb HH-2, where the immunogenicity and safety of a heterologous booster with PHH-1V is assessed versus a homologous booster with BNT162b2 at 14, 28 and 98 days after vaccine administration. Methods: The HH-2 study is an ongoing multicentre, randomised, active-controlled, double-blind, non-inferiority Phase IIb trial, where participants 18 years or older who had received two doses of BNT162b2 were randomly assigned in a 2:1 ratio to receive a booster dose of vaccine-either heterologous (PHH-1V group) or homologous (BNT162b2 group)-in 10 centres in Spain. Eligible subjects were allocated to treatment stratified by age group (18-64 versus ≥65 years) with approximately 10% of the sample enrolled in the older age group. The primary endpoints were humoral immunogenicity measured by changes in levels of neutralizing antibodies (PBNA) against the ancestral Wuhan-Hu-1 strain after the PHH-1V or the BNT162b2 boost, and the safety and tolerability of PHH-1V as a boost. The secondary endpoints were to compare changes in levels of neutralizing antibodies against different variants of SARS-CoV-2 and the T-cell responses towards the SARS-CoV-2 spike glycoprotein peptides. The exploratory endpoint was to assess the number of subjects with SARS-CoV-2 infections ≥14 days after PHH-1V booster. This study is ongoing and is registered with ClinicalTrials.gov, NCT05142553. Findings: From 15 November 2021, 782 adults were randomly assigned to PHH-1V (n = 522) or BNT162b2 (n = 260) boost vaccine groups. The geometric mean titre (GMT) ratio of neutralizing antibodies on days 14, 28 and 98, shown as BNT162b2 active control versus PHH-1V, was, respectively, 1.68 (p < 0.0001), 1.31 (p = 0.0007) and 0.86 (p = 0.40) for the ancestral Wuhan-Hu-1 strain; 0.62 (p < 0.0001), 0.65 (p < 0.0001) and 0.56 (p = 0.003) for the Beta variant; 1.01 (p = 0.92), 0.88 (p = 0.11) and 0.52 (p = 0.0003) for the Delta variant; and 0.59 (p ≤ 0.0001), 0.66 (p < 0.0001) and 0.57 (p = 0.0028) for the Omicron BA.1 variant. Additionally, PHH-1V as a booster dose induced a significant increase of CD4+ and CD8+ T-cells expressing IFN-γ on day 14. There were 458 participants who experienced at least one adverse event (89.3%) in the PHH-1V and 238 (94.4%) in the BNT162b2 group. The most frequent adverse events were injection site pain (79.7% and 89.3%), fatigue (27.5% and 42.1%) and headache (31.2 and 40.1%) for the PHH-1V and the BNT162b2 groups, respectively. A total of 52 COVID-19 cases occurred from day 14 post-vaccination (10.14%) for the PHH-1V group and 30 (11.90%) for the BNT162b2 group (p = 0.45), and none of the subjects developed severe COVID-19. Interpretation: Our interim results from the Phase IIb HH-2 trial show that PHH-1V as a heterologous booster vaccine, when compared to BNT162b2, although it does not reach a non-inferior neutralizing antibody response against the Wuhan-Hu-1 strain at days 14 and 28 after vaccination, it does so at day 98. PHH-1V as a heterologous booster elicits a superior neutralizing antibody response against the previous circulating Beta and the currently circulating Omicron BA.1 SARS-CoV-2 variants in all time points assessed, and for the Delta variant on day 98 as well. Moreover, the PHH-1V boost also induces a strong and balanced T-cell response. Concerning the safety profile, subjects in the PHH-1V group report significantly fewer adverse events than those in the BNT162b2 group, most of mild intensity, and both vaccine groups present comparable COVID-19 breakthrough cases, none of them severe. Funding: HIPRA SCIENTIFIC, S.L.U.

Risk Factors for COVID-19 in Inflammatory Bowel Disease: A National, ENEIDA-Based Case-Control Study (COVID-19-EII).

(1) Scant information is available concerning the characteristics that may favour the acquisition of COVID-19 in patients with inflammatory bowel disease (IBD). Therefore, the aim of this study was to assess these differences between infected and noninfected patients with IBD. (2) This nationwide case−control study evaluated patients with inflammatory bowel disease with COVID-19 (cases) and without COVID-19 (controls) during the period March−July 2020 included in the ENEIDA of GETECCU. (3) A total of 496 cases and 964 controls from 73 Spanish centres were included. No differences were found in the basal characteristics between cases and controls. Cases had higher comorbidity Charlson scores (24% vs. 19%; p = 0.02) and occupational risk (28% vs. 10.5%; p < 0.0001) more frequently than did controls. Lockdown was the only protective measure against COVID-19 (50% vs. 70%; p < 0.0001). No differences were found in the use of systemic steroids, immunosuppressants or biologics between cases and controls. Cases were more often treated with 5-aminosalicylates (42% vs. 34%; p = 0.003). Having a moderate Charlson score (OR: 2.7; 95%CI: 1.3−5.9), occupational risk (OR: 2.9; 95%CI: 1.8−4.4) and the use of 5-aminosalicylates (OR: 1.7; 95%CI: 1.2−2.5) were factors for COVID-19. The strict lockdown was the only protective factor (OR: 0.1; 95%CI: 0.09−0.2). (4) Comorbidities and occupational exposure are the most relevant factors for COVID-19 in patients with IBD. The risk of COVID-19 seems not to be increased by immunosuppressants or biologics, with a potential effect of 5-aminosalicylates, which should be investigated further and interpreted with caution.

Immune response and reactogenicity after immunization with two-doses of an experimental COVID-19 vaccine (CVnCOV) followed by a third-fourth shot with a standard mRNA vaccine (BNT162b2): RescueVacs multicenter cohort study.

Background: There is no evidence to date on immunogenic response among individuals who participated in clinical trials of COVID-19 experimental vaccines redirected to standard national vaccination regimens. Methods: This multicentre, prospective controlled cohort study included subjects who received a COVID-19 experimental vaccine (CVnCoV)(test group, TG) - and unvaccinated subjects (control group, CG), selected among individuals to be vaccinated according to the Spanish vaccination program. All study subjects received BNT162b2 as a standard national vaccination schedule, except 8 (from CG) who received mRNA-1273 and were excluded from immunogenicity analyses. Anti-RBD antibodies level and neutralising titres (NT50) against G614, Beta, Mu, Delta and Omicron variants were analysed. Reactogenicity was also assessed. Findings: 130 participants (TG:92; CG:38) completed standard vaccination. In TG, median (IQR) of anti-RBD antibodies after first BNT162b2 dose were 10740·0 BAU/mL (4466·0-12500) compared to 29·8 BAU/mL (14·5-47·8) in CG (p <0·0001). Median NT50 (IQR) of G614 was 2674·0 (1865·0-3997·0) in TG and 63·0 (16·0-123·1) in CG (p <0·0001). After second BNT162b2 dose, anti-RBD levels increased to ≥12500 BAU/mL (11625·0-12500) in TG compared to 1859·0 BAU/mL (915·4-3820·0) in CG (p <0·0001). NT50 was 2626·5 (1756·0-5472·0) and 850·4 (525·1-1608·0), respectively (p <0·0001). Variant-specific (Beta, Mu, Omicron) response was also assessed. Most frequent adverse reactions were headache, myalgia, and local pain. No severe AEs were reported. Interpretation: Heterologous BNT162b2 as third and fourth doses in previously suboptimal immunized individuals elicit stronger immune response than that obtained with two doses of BNT162b2. This apparent benefit was also observed in variant-specific response. No safety concerns arose. Funding: Partly funded by the Institute of Health Carlos-III and COVID-19 Fund, co-financed by the European Regional Development Fund (FEDER) "A way to make Europe".

Nationwide COVID-19-EII Study: Incidence, Environmental Risk Factors and Long-Term Follow-Up of Patients with Inflammatory Bowel Disease and COVID-19 of the ENEIDA Registry.

We aim to describe the incidence and source of contagion of COVID-19 in patients with IBD, as well as the risk factors for a severe course and long-term sequelae. This is a prospective observational study of IBD and COVID-19 included in the ENEIDA registry (53,682 from 73 centres) between March-July 2020 followed-up for 12 months. Results were compared with data of the general population (National Centre of Epidemiology and Catalonia). A total of 482 patients with COVID-19 were identified. Twenty-eight percent were infected in the work environment, and 48% were infected by intrafamilial transmission, despite having good adherence to lockdown. Thirty-five percent required hospitalization, 7.9% had severe COVID-19 and 3.7% died. Similar data were reported in the general population (hospitalisation 19.5%, ICU 2.1% and mortality 4.6%). Factors related to death and severe COVID-19 were being aged ≥ 60 years (OR 7.1, 95% CI: 1.8-27 and 4.5, 95% CI: 1.3-15.9), while having ≥2 comorbidities increased mortality (OR 3.9, 95% CI: 1.3-11.6). None of the drugs for IBD were related to severe COVID-19. Immunosuppression was definitively stopped in 1% of patients at 12 months. The prognosis of COVID-19 in IBD, even in immunosuppressed patients, is similar to that in the general population. Thus, there is no need for more strict protection measures in IBD.

Ovarian stimulated cycles reduce protection of follicular fluid against free radicals.

Controlled ovarian hyperstimulation cycle with exogenous gonadotropins (COH) is associated with clinical complications. The aim of this work was to determine whether COH alters the physiological antioxidant status of follicular fluid in women with no reproductive dysfunction, compared to the natural cycle (NC). In this longitudinal study, forty-one women (oocyte donors) consecutively underwent NC and COH. Follicular fluid was collected at oocyte retrieval and different redox biomarkers were determined: total antioxidant activity (TAA), oxygen radical absorbance capacity (ORAC), nitric oxide, α- and γ-tocopherol, the fatty acid composition, activities of superoxide dismutase, catalase, total and Se-dependent glutathione peroxidases, and the antioxidant paraoxonase (PON) family. Results showed that TAA (1.70 ±â€¯0.03 mM versus 1.86 ±â€¯0.03 mM, p < 0.05), α-tocopherol (4.37 ±â€¯0.26 µM versus 5.74 ±â€¯0.30 µM, p < 0.05), PON1 paraoxonase (245 ±â€¯24 nmol/min/ml versus 272 ±â€¯27 nmol/min/ml, p < 0.05), PON1 arylesterase (87.2 ±â€¯4.6 µmol/min/ml versus 99.3 ±â€¯4.8 µmol/min/ml, p < 0.05), and PON3 simvastatinase (13.48 ±â€¯0.52 nmol/min/ml versus 16.29 ±â€¯0.72 nmol/min/ml, p < 0.001) were significantly lower in COH versus NC. Fatty acids from COH were more saturated, increasing palmitate and decreasing the n-6 and total polyunsaturated fatty acids (PUFAs). Docosahexaenoic acid also increased (p < 0.05). Results suggest that COH could lead to premature ovarian aging and provide new insights into the possible prevention of the adverse effects of ovarian hyperstimulation by directing therapeutic applications to the maintenance of the redox balance and fatty acid status, with special attention to paraoxonase proteins and docosahexaenoic acid.

Lower follicular n-3 polyunsaturated fatty acid levels are associated with a better response to ovarian stimulation.

OBJECTIVES: To analyze in detail the fatty acid (FA) composition of follicular fluid (FF) from two-sized follicles at oocyte retrieval and to determine associations of the FAs from large follicles with the woman's age and the response to ovarian stimulation. DESIGN: Observational study. SETTING: University and fertility clinic. PATIENTS: Sixty-four women (age 19-46), consisting of unfertile patients and oocyte donors, undergoing controlled ovarian stimulation. INTERVENTIONS: None. MAIN OUTCOME MEASURE(S): FF from small (< 12 mm) and large (≥ 18 mm) follicles was collected at oocyte retrieval. FAs by gas chromatography-flame ionization detection. RESULT: Thirty-two FAs with chain lengths ranging from 14 to 25 carbons were identified. There was a readjustment in FA distribution as follicle size increased, raising very long-chain saturated FAs, nervonic (24:1n-9), arachidonic (20:4n-6), and n-3 polyunsaturated FAs (PUFA, P < 0.001), the latter mainly due to an increase in docosahexaenoic acid (22:6n-3, DHA). In large follicles, double bond and peroxidizability indices and total n-3 PUFA, particularly DHA, correlated positively with the woman's age and negatively with the number of total and mature oocytes, total and top-quality embryos, and fertilization rate. CONCLUSIONS: We have described 32 FAs in ovarian FF, of which 16 changed their distribution with follicle size. The results also indicate that lower n-3 PUFA levels in large follicles, which are associated with younger women, predict a better response to ovarian stimulation based on the recovery of total and mature oocytes, total and top-quality embryos, and fertilization rate per cycle. KEY MESSAGE: The fatty acid profile of ovarian FF changes as the follicle grows and lower n-3 PUFA levels in large follicles, associated with younger women, predict a better response to ovarian stimulation.

Analysis of Protein Oxidative Modifications in Follicular Fluid from Fertile Women: Natural Versus Stimulated Cycles.

Oxidative stress is associated with obstetric complications during ovarian hyperstimulation in women undergoing in vitro fertilization. The follicular fluid contains high levels of proteins, which are the main targets of free radicals. The aim of this work was to determine specific biomarkers of non-enzymatic oxidative modifications of proteins from follicular fluid in vivo, and the effect of ovarian stimulation with gonadotropins on these biomarkers. For this purpose, 27 fertile women underwent both a natural and a stimulated cycle. The biomarkers, glutamic semialdehyde (GSA), aminoadipic semialdehyde (AASA), Nε-(carboxymethyl)lysine (CML), and Nε-(carboxyethyl)lysine (CEL), were measured by gas-liquid chromatography coupled to mass spectrometry. Results showed that follicular fluid contained products of protein modifications by direct metal-catalyzed oxidation (GSA and AASA), glycoxidation (CML and CEL), and lipoxidation (CML). GSA was the most abundant biomarker (91.5%). The levels of CML amounted to 6% of the total lesions and were higher than AASA (1.3%) and CEL (1.2%). In the natural cycle, CEL was significantly lower (p < 0.05) than in the stimulated cycle, suggesting that natural cycles are more protected against protein glycoxidation. These findings are the basis for further research to elucidate the possible relevance of this follicular biomarker of advanced glycation end product in fertility programs.

Paraoxonase activities in human follicular fluid: role in follicular maturation.

The paraoxonases (PONs) are antioxidant enzymes associated with beneficial effects against several diseases and some exposures. Little is known, however, about the role of PONs in human reproduction. This work was conducted to investigate whether any association existed between the activities of the PON enzymes (1, 2, and 3) with the follicular size and fertility parameters in assisted reproduction. The study included 100 subfertile women (patients) and 55 proven fertile women (oocyte donors), all undergoing an ovarian stimulation cycle. Follicular fluid from small (diameter <12 mm) and large (diameter ≥18 mm) follicles was collected from each woman. The PONs were quantified in follicular fluid by immunoblotting. PON1 arylesterase and paraoxonase, PON2 methyl paraoxonase and PON3 simvastatinase activities from both donors and patients were significantly higher (P < 0.001) in follicular fluid from large follicles compared with small ones. In large follicles, PON3 activity was significantly higher (P < 0.01) in donors compared with patients. Follicular fluid PON1 arylesterase and paraoxonase activity was positively correlated with the number of retrieved oocytes in donors. This study shows an increase in the activities of PONs with follicle size, thus providing indirect evidence for the role of PONs in follicle maturation.

Influence of oxygen partial pressure on the characteristics of human hepatocarcinoma cells.

Most of the in vitro studies using liver cell lines have been performed under atmospheric oxygen partial pressure (21% O2). However, the oxygen concentrations in the liver and cancer cells are far from this value. In the present study, we have evaluated the influence of oxygen on 1) the tumor cell lines features (growth, steady-state ROS levels, GSH content, activities of antioxidant enzymes, p66 Shc and SOD expressions, metalloproteinases secretion, migration, invasion, and adhesion) of human hepatocellular carcinoma cell lines, and b) the response of the cells to an oxidant stimulus (aqueous leaf extract of the V. baccifera plant species). For this purpose, three hepatocarcinoma cell lines with different p53 status, HepG2 (wild-type), Huh7 (mutated), and Hep3B (deleted), were cultured (6-30 days) under atmospheric (21%) and more physiological (8%) pO2. Results showed that after long-term culturing at 8% versus 21% O2, the cellular proliferation rate and the steady-state levels of mitochondrial O2- were unaffected. However, the intracellular basal ROS levels were higher independently of the characteristics of the cell line. Moreover, the lower pO2 was associated with lower glutathione content, the induction of p66 Shc and Mn-SOD proteins, and increased SOD activity only in HepG2. This cell line also showed a higher migration rate, secretion of active metalloproteinases, and a faster invasion. HepG2 cells were more resistant to the oxidative stress induced by V. baccifera. Results suggest that the long-term culturing of human hepatoma cells at a low, more physiological pO2 induces antioxidant adaptations that could be mediated by p53, and may alter the cellular response to a subsequent oxidant challenge. Data support the necessity of validating outcomes from studies performed with hepatoma cell cultures under ambient O2.

Glutathione S-transferase activity in follicular fluid from women undergoing ovarian stimulation: role in maturation.

Female infertility involves an emotional impact for the woman, often leading to a state of anxiety and low self-esteem. The assisted reproduction techniques (ART) are used to overcome the problem of infertility. In a first step of the in vitro fertilization therapy women are subjected to an ovarian stimulation protocol to obtain mature oocytes, which will result in competent oocytes necessary for fertilization to occur. Ovarian stimulation, however, subjects the women to a high physical and psychological stress, thus being essential to improve ART and to find biomarkers of dysfunction and fertility. GSH is an important antioxidant, and is also used in detoxification reactions, catalysed by glutathione S-transferases (GST). In the present work, we have investigated the involvement of GST in follicular maturation. Patients with fertility problems and oocyte donors were recruited for the study. From each woman follicles at two stages of maturation were extracted at the preovulatory stage. Follicular fluid was separated from the oocyte by centrifugation and used as the enzyme source. GST activity was determined based on its conjugation with 3,4-dichloronitrobenzene and the assay was adapted to a 96-well microplate reader. The absorbance was represented against the incubation time and the curves were adjusted to linearity (R(2)>0.990). Results showed that in both donors and patients GST activity was significantly lower in mature oocytes compared to small ones. These results suggest that GST may play a role in the follicle maturation by detoxifying xenobiotics, thus contributing to the normal development of the oocyte. Supported by FIS/FEDER (PI11/02559), Gobierno Vasco (Dep. Educación, Universiades e Investigación, IT687-13), and UPV/EHU (CLUMBER UFI11/20 and PES13/58). The work was approved by the Ethics Committee of the UPV/EHU (CEISH/96/2011/RUIZLARREA), and performed according to the UPV/EHU and IVI-Bilbao agreement (Ref. 2012/01).

Characterization of the paraoxonase system in follicular fluid of women subjected to an ovarian stimulation cycle.

Reactive oxygen species (ROS) and antioxidants are involved in the regulation of reproductive processes. Previous studies in infertile women undergoing an ovarian stimulation cycle have suggested a possible role for ROS in the occurrence of conception in In Vitro Fertilization. In this context the control of the redox balance of follicular fluid becomes essential for reproduction, so that the presence of enzymes with antioxidant activities, such as the paraoxonase (PON) system, would play a role in maintaining this balance. The objective of this work was a) to characterize the paraoxonase system in follicular fluid of women undergoing a controlled ovarian stimulation cycle, analysing the associated PON1, PON2, and PON3 activities, and b) to study the possible involvement of the PON system in follicular maturation. The enzyme activities were quantified in follicular fluid from large and small follicles from women undergoing an ovarian stimulation cycle in the IVI-Bilbao clinic. PON activities were quantified using spectrophotometric and HPLC techniques. Statistical comparisons were performed using the Student's t-test for paired data. Results indicate that follicular fluid presents paraoxonase activities which are detectable by the methods developed in this study. PON activities were associated with follicular maturation, suggesting that the PON system plays a role in oocyte maturation. This work was supported by research grants from the Ministry of Health and Consumption (FIS/FEDER PI11/02559), the Basque Country Government (Dep. Education, Universities and Research ref. IT687-13, and DCIT ref. S-PE13UN063), and UPV/EHU (CLUMBER UFI11/20 and PES13/58). The work was approved by the Ethics Committee of the UPV/EHU (CEISH/96/2011/RUIZLARREA), and performed according to the UPV/EHU and IVI-Bilbao agreement (Ref. 2012/01).