A peptide derived from HSP60 reduces proinflammatory cytokines and soluble mediators: a therapeutic approach to inflammation.

Cytokines are secretion proteins that mediate and regulate immunity and inflammation. They are crucial in the progress of acute inflammatory diseases and autoimmunity. In fact, the inhibition of proinflammatory cytokines has been widely tested in the treatment of rheumatoid arthritis (RA). Some of these inhibitors have been used in the treatment of COVID-19 patients to improve survival rates. However, controlling the extent of inflammation with cytokine inhibitors is still a challenge because these molecules are redundant and pleiotropic. Here we review a novel therapeutic approach based on the use of the HSP60-derived Altered Peptide Ligand (APL) designed for RA and repositioned for the treatment of COVID-19 patients with hyperinflammation. HSP60 is a molecular chaperone found in all cells. It is involved in a wide diversity of cellular events including protein folding and trafficking. HSP60 concentration increases during cellular stress, for example inflammation. This protein has a dual role in immunity. Some HSP60-derived soluble epitopes induce inflammation, while others are immunoregulatory. Our HSP60-derived APL decreases the concentration of cytokines and induces the increase of FOXP3+ regulatory T cells (Treg) in various experimental systems. Furthermore, it decreases several cytokines and soluble mediators that are raised in RA, as well as decreases the excessive inflammatory response induced by SARS-CoV-2. This approach can be extended to other inflammatory diseases.

Evidence of SARS-CoV-2 infection in postmortem lung, kidney, and liver samples, revealing cellular targets involved in COVID-19 pathogenesis.

There is an urgent need to understand severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-host interactions involved in virus spread and pathogenesis, which might contribute to the identification of new therapeutic targets. In this study, we investigated the presence of SARS-CoV-2 in postmortem lung, kidney, and liver samples of patients who died with coronavirus disease (COVID-19) and its relationship with host factors involved in virus spread and pathogenesis, using microscopy-based methods. The cases analyzed showed advanced stages of diffuse acute alveolar damage and fibrosis. We identified the SARS-CoV-2 nucleocapsid (NC) in a variety of cells, colocalizing with mitochondrial proteins, lipid droplets (LDs), and key host proteins that have been implicated in inflammation, tissue repair, and the SARS-CoV-2 life cycle (vimentin, NLRP3, fibronectin, LC3B, DDX3X, and PPARγ), pointing to vimentin and LDs as platforms involved not only in the viral life cycle but also in inflammation and pathogenesis. SARS-CoV-2 isolated from a patient´s nasal swab was grown in cell culture and used to infect hamsters. Target cells identified in human tissue samples included lung epithelial and endothelial cells; lipogenic fibroblast-like cells (FLCs) showing features of lipofibroblasts such as activated PPARγ signaling and LDs; lung FLCs expressing fibronectin and vimentin and macrophages, both with evidence of NLRP3- and IL1ß-induced responses; regulatory cells expressing immune-checkpoint proteins involved in lung repair responses and contributing to inflammatory responses in the lung; CD34+ liver endothelial cells and hepatocytes expressing vimentin; renal interstitial cells; and the juxtaglomerular apparatus. This suggests that SARS-CoV-2 may directly interfere with critical lung, renal, and liver functions involved in COVID-19-pathogenesis.

Jusvinza, an anti-inflammatory drug derived from the human heat-shock protein 60, for critically ill COVID-19 patients. An observational study.

This paper presents the results of an observational and retrospective study on the therapeutic effects of Jusvinza, an immunomodulatory peptide with anti-inflammatory properties for critically ill COVID-19 patients. This peptide induces regulatory mechanisms on the immune response in experimental systems and in patients with Rheumatoid Arthritis. Exploratory research in COVID-19 patients revealed that Jusvinza promotes clinical and radiological improvement. The aim of this study is to describe the clinical outcome and variations of several inflammatory biomarkers in a cohort of critically ill COVID-19 patients, divided into two groups during the observational research: one group received Jusvinza and the other did not. Research physicians extracted the patients´ data from their hospital's clinical records. The study analyzed 345 medical records, and 249 records from critically ill patients were included. The data covered the demographic characteristics, vital signs, ventilatory parameters and inflammatory biomarkers. Survival outcome was significantly higher in the group receiving Jusvinza (90.4%) compared to the group without Jusvinza (39.5%). Furthermore, in patients treated with Jusvinza there was a significant improvement in ventilatory parameters and a reduction in inflammation and coagulation biomarkers. Our findings show that Jusvinza could control the extent of inflammation in COVID-19 patients. This study indicates that Jusvinza is a helpful drug for the treatment of diseases characterized by hyperinflammation.

Correction to "Cuban Abdala vaccine: Effectiveness in preventing severe disease and death from COVID-19 in Havana, Cuba; A cohort study" [The Lancet Regional Health - Americas 2022; 16: 100366] https://doi.org/10.1016/j.lana.2022.100366.

[This corrects the article DOI: 10.1016/j.lana.2022.100366.].

An engineered SARS-CoV-2 receptor-binding domain produced in Pichia pastoris as a candidate vaccine antigen.

Developing affordable and easily manufactured SARS-CoV-2 vaccines will be essential to achieve worldwide vaccine coverage and long-term control of the COVID-19 pandemic. Here the development is reported of a vaccine based on the SARS-CoV-2 receptor-binding domain (RBD), produced in the yeast Pichia pastoris. The RBD was modified by adding flexible N- and C-terminal amino acid extensions that modulate protein/protein interactions and facilitate protein purification. A fed-batch methanol fermentation with a yeast extract-based culture medium in a 50 L fermenter and an immobilized metal ion affinity chromatography-based downstream purification process yielded 30-40 mg/L of RBD. Correct folding of the purified protein was demonstrated by mass spectrometry, circular dichroism, and determinations of binding affinity to the angiotensin-converting enzyme 2 (ACE2) receptor. The RBD antigen also exhibited high reactivity with sera from convalescent individuals and Pfizer-BioNTech or Sputnik V vaccinees. Immunization of mice and non-human primates with 50 µg of the recombinant RBD adjuvanted with alum induced high levels of binding antibodies as assessed by ELISA with RBD produced in HEK293T cells, and which inhibited RBD binding to ACE2 and neutralized infection of VeroE6 cells by SARS-CoV-2. Additionally, the RBD protein stimulated IFNγ, IL-2, IL-6, IL-4 and TNFα secretion in splenocytes and lung CD3+-enriched cells of immunized mice. The data suggest that the RBD recombinant protein produced in yeast P. pastoris is suitable as a vaccine candidate against COVID-19.

Cuban Abdala vaccine: Effectiveness in preventing severe disease and death from COVID-19 in Havana, Cuba; A cohort study.

Background: COVID-19 vaccines have proven safe and efficacious in reducing severe illness and death. Cuban protein subunit vaccine Abdala has shown safety, tolerability and efficacy (92·3% [95% CI: 85·7‒95·8]) against SARS-CoV-2 in clinical trials. This study aimed to estimate Abdala's real-world vaccine effectiveness (VE). Methods: This retrospective cohort study in Havana analyzed Cuban Ministry of Public Health databases (May 12-August 31, 2021) to assess VE in preventing severe illness and death from COVID-19 (primary outcomes). Cox models accounting for time-varying vaccination status and adjusting by demographics were used to estimate hazard ratios. A subgroup analysis by age group and a sensitivity analysis including a subgroup of tested persons (qRT-PCR) were conducted. Daily cases and deaths were modelled accounting for different VE. Findings: The study included 1 355 638 persons (Mean age: 49·5 years [SD: 18·2]; 704 932 female [52·0%]; ethnicity data unavailable): 1 324 vaccinated (partially/fully) and 31 433 unvaccinated. Estimated VE against severe illness was 93·3% (95% CI: 92·1-94·3) in partially- vaccinated and 98·2% (95% CI: 97·9-98·5) in fully-vaccinated and against death was 94·1% (95% CI: 92·5-95·4) in partially-vaccinated and 98·7% (95% CI: 98·3-99·0) in fully-vaccinated. VE exceeded 92·0% in all age groups. Daily cases and deaths during the study period corresponded to a VE above 90%, as predicted by models. Interpretation: The Cuban Abdala protein subunit vaccine was highly effective in preventing severe illness and death from COVID-19 under real-life conditions. Funding: Cuban Ministry of Public Health. Genetic Engineering and Biotechnology Centre.

Safety, tolerability, and immunogenicity of a SARS-CoV-2 recombinant spike RBD protein vaccine: A randomised, double-blind, placebo-controlled, phase 1-2 clinical trial (ABDALA Study).

Background: Multiple vaccine candidates against COVID-19 are currently being evaluated. We evaluate the safety and immunogenicity protein of a novel SARS-CoV-2 virus receptor-binding domain (RBD) vaccine. Methods: A phase 1-2, randomised, double-blind, placebo-controlled trial was carried out in "Saturnino Lora" Hospital, Santiago de Cuba, Cuba. Subjects (healthy or those with controlled chronic diseases) aged between 19 and 80 years, who gave written informed consent were eligible. Subjects were randomly assigned (1:1:1, in blocks) to three groups: placebo, 25 µg and 50 µg RBD vaccine (Abdala). The product was administered intramuscularly, 0·5 mL in the deltoid region. During the first phase, two immunization schedules were studied: 0-14-28 days (short) and 0-28-56 days (long). In phase 2, only the short schedule was evaluated. The organoleptic characteristics and presentations of vaccine and placebo were identical. All participants (subjects, clinical researchers, statisticians, laboratory technicians, and monitors) remained masked during the study period. The main endpoints were safety and the proportion of subjects with seroconversion of anti-RBD IgG antibodies, analysed by intention to treat and per protocol, respectively. The trial is registered with the Cuban Public Registry of Clinical Trials, RPCEC00000346. Findings: Between Dec 7, 2020, and Feb 9, 2021, 792 subjects were included; 132 (66 in each vaccination schedule, divided into 22 for each group) in phase 1, and 660 (220 in each group plus 66 from the short scheme of phase 1) in phase 2. The product was well tolerated. No severe adverse events were reported. During phase 1, the incidence of adverse events in the 25 µg, 50 µg, and placebo arms for the short schedule were 6/22 (27·3%), 6/22 (27·3%), 3/22 (13·6%), respectively, and for the long schedule were 8/22 (36·4%), 9/22 (40·9%), 4/22 (18·2%), respectively. In phase 2, adverse reactions were reported by 53/242 (21·9%), 75/242 (31·0%) and 41/242 (16·9%) participants in the 25 µg, 50 µg, and placebo group, respectively. Adverse reactions were minimal, mostly mild, and from the injection site, which resolved in the first 24-48 hours. In phase 1, seroconversion at day 56 was seen in 95·2% of the participants (20/21) in the 50 µg group, 81% (17/21) in the 25 µg group, and none in the placebo group (0/22). For the long schedule, seroconversion at day 70 was seen in 100% of the participants (21/21) in the 50 µg group, 94·7% (18/19) in the 25 µg group, and none in the placebo group (0/22). In phase 2, seroconversion of anti-RBD IgG antibodies at day 56 was seen in 89·2% of the participants in the 50 µg group (214/240; 95% CI 84·5-92·82), 77·7% in the 25 µg group (185/238; 72·0-82·9) and 4·6% in the placebo group (11/239; 2·3-8·1). Compared with the placebo arm, the differences in the proportion of participants with seroconversion were 73·1% (95% CI 66·8-79·5) and 84·6% (79·4-89·7) in the 25 µg and 50 µg groups, respectively. The seroconversion rate in the 50 µg group was significantly higher than in the 25 µg group (p=0·0012). Interpretation: The Abdala vaccine was safe, well tolerated, and induced humoral immune responses against SARS-CoV-2. These results, in the context of the emergency COVID-19 pandemic, support the 50 µg dose, applied in a 0-14-28 days schedule, for further clinical trials to confirm vaccine efficacy. Funding: Centre for Genetic Engineering and Biotechnology (CIGB), Havana, Cuba.

The microglial NLRP3 inflammasome is involved in human SARS-CoV-2 cerebral pathogenicity: A report of three post-mortem cases.

We herein report, by using confocal immunofluorescence, the colocalization of the SARS-CoV-2 nucleocapsid within neurons, astrocytes, oligodendrocytes and microglia in three deceased COVID-19 cases, of between 78 and 85 years of age at death. The viral nucleocapsid was detected together with its ACE2 cell entry receptor, as well as the NLRP3 inflammasome in cerebral cortical tissues. It is noteworthy that NLRP3 was colocalized with CD68 + macrophages in the brain and lung of the deceased, suggesting the critical role of this type of inflammasome in SARS-CoV-2 lesions of the nervous system/lungs and supporting its potential role as a therapeutic target.

Repurposing NASVAC, a hepatitis B therapeutic vaccine, for pre- and postexposure prophylaxis of SARS-CoV-2 infection (preprint)

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the subsequent coronavirus 2019 (COVID-19) have led to tens of millions of cases and millions of deaths around the world. Although more than a year has passed since the emergence of COVID-19, more waves of the pandemic, with new variants of the deadly virus, have been reported. It seems that the virus will continue to infect people for years or decades to come and thus lead to more illnesses and deaths. The experiences last year regarding limiting the transmission of the virus indicate that one or more traditional methods of containment may not be effective; further, even vaccination may not give immunity to society. On the other hand, eliminating the virus using drugs capable of eradicating SARS-CoV-2 from the infected host may not be an achievable goal. Based on these realities and after exploring the mechanism underlying the acquisition of the virus and pathogenesis of COVID-19, we assumed that immune therapy may be a practical option for the containment of SARS-CoV-2. In this study, we repurposed an immune modulator containing two antigens of hepatitis B virus, hepatitis B surface antigen (HBsAg) and hepatitis B core antigen (HBcAg) (termed NASVAC, Center for Genetic Engineering and Biotechnology, CIGB, Havana, Cuba), to gain insight into its role against SARS-CoV-2. NASVAC induced cytokines of innate immunity following nasal administration and prevented all 20 volunteers from being diagnosed with SARS-CoV-2 during the two weeks of usage. Four volunteers were infected with SARS-CoV-2 two weeks after the end of NASVAC administration; three of them showed almost no symptoms and recovered without any intervention, and one with several comorbidities attended a hospital for four days and recovered completely. In conclusion, the administration of NASVAC to subjects at risk of SARS-CoV-2 infection was safe. The pattern of cytokine responses and absence of infection or mild COVID-19 infection of the subjects involved in the study are preliminary evidence indicating that this product may prevent or suppress SARS-CoV-2 infection at the initial stages of SARS-CoV-2 acquisition and/or replication and deserve further exploratory trials to confirm the capacity of NASVAC as pre/postexposure prophylaxis or pre-emptive therapy in the context of SARS-CoV-2 infection.

Revisiting Pleiotropic Effects of Type I Interferons: Rationale for Its Prophylactic and Therapeutic Use Against SARS-CoV-2.

The pandemic distribution of SARS-CoV-2 together with its particular feature of inactivating the interferon-based endogenous response and accordingly, impairing the innate immunity, has become a challenge for the international scientific and medical community. Fortunately, recombinant interferons as therapeutic products have accumulated a long history of beneficial therapeutic results in the treatment of chronic and acute viral diseases and also in the therapy of some types of cancer. One of the first antiviral treatments during the onset of COVID-19 in China was based on the use of recombinant interferon alfa 2b, so many clinicians began to use it, not only as therapy but also as a prophylactic approach, mainly in medical personnel. At the same time, basic research on interferons provided new insights that have contributed to a much better understanding of how treatment with interferons, initially considered as antivirals, actually has a much broader pharmacological scope. In this review, we briefly describe interferons, how they are induced in the event of a viral infection, and how they elicit signaling after contact with their specific receptor on target cells. Additionally, some of the genes stimulated by type I interferons are described, as well as the way interferon-mediated signaling is torpedoed by coronaviruses and in particular by SARS-CoV-2. Angiotensin converting enzyme 2 (ACE2) gene is one of the interferon response genes. Although for many scientists this fact could result in an adverse effect of interferon treatment in COVID-19 patients, ACE2 expression contributes to the balance of the renin-angiotensin system, which is greatly affected by SARS-CoV-2 in its internalization into the cell. This manuscript also includes the relationship between type I interferons and neutrophils, NETosis, and interleukin 17. Finally, under the subtitle of "take-home messages", we discuss the rationale behind a timely treatment with interferons in the context of COVID-19 is emphasized.

Effect and safety of combination of interferon alpha-2b and gamma or interferon alpha-2b for negativization of SARS-CoV-2 viral RNA. Preliminary results of a randomized controlled clinical trial. (preprint)

Abstract Objectives: IFN-alpha2b and IFN-gamma combination has demonstrated favorable pharmacodynamics for genes underlying antiviral activity which might be involved in the defense of the organism from a SARS-CoV-2 infection. Considering this we conducted a randomized controlled clinical trial for efficacy and safety evaluation of subcutaneous IFN-alpha2b and IFN-gamma administration in patients positive to SARS-CoV-2. Methods: We enrolled 19-82 years-old inpatients at the Military Central Hospital Luis Diaz Soto, Havana, Cuba. They were hospitalized after confirmed diagnosis for SARS-CoV-2 RNA by real-time reverse transcription polymerase chain reaction. Patients were randomly assigned in a 1:1 ratio to receive either, subcutaneous treatment with a co-lyophilized combination of 3.0 MIU IFN-alpha2b and 0.5 MIU IFN-gamma (HeberFERON, CIGB, Havana, Cuba), twice a week for two weeks, or thrice a week intramuscular injection of 3.0 MIU IFN-alpha2b (Heberon Alpha R, CIGB, Havana, Cuba). Additionally, all patients received lopinavir-ritonavir 200/50 mg every 12 h and chloroquine 250 mg every 12 h (standard of care). The primary endpoints were the time to negativization of viral RNA and the time to progression to severe COVID-19, from the start of treatment. The protocol was approved by the Ethics Committee on Clinical Investigation from the Hospital and the Center for the State Control of Medicines, Equipment and Medical Devices in Cuba. Informed consent was obtained from each participant. Results: A total of 79 patients with laboratory-confirmed SARS-CoV-2 infection, including symptomatic or asymptomatic conditions, fulfilled the inclusion criteria and underwent randomization. Thirty-three subjects were assigned to the HeberFERON group, and 33 to the Heberon Alpha R group. Sixty-three patients were analyzed for viral negativization, of them 78.6% in the HeberFERON group negativized the virus after 4 days of treatment versus 40.6% of patients in the Heberon Alpha R groups (p=0.004). Time to reach the negativization of the SARS-CoV-2 measured by RT-PCR in real time was of 3.0 and 5.0 days for the HeberFERON and Heberon Alpha R groups, respectively. A significant improvement in the reduction of time for negativization was attributable to HeberFERON (p=0.0027, Log-rank test) with a Hazard Ratio of 3.2 and 95% CI of 1.529 to 6.948, as compared to Heberon Alpha R treated group. Worsening of respiratory symptoms was detected in two (6.6%) and one (3.3%) patients in HeberFERON and IFN-alpha2b groups, respectively. None of the subjects transit to severe COVID-19 during the study or the epidemiological follow-up for 21 more days. RT-PCR on day 14 after the start of the treatment was negative to SARS-CoV-2 in 100% and 91% of patients of the combination of IFNs and IFN-alpha2b, respectively. Negativization for HeberFERON treated patients was related to a significant increase in lymphocytes counts and an also significant reduction in CRP as early as 7 days after commencing the therapeutic schedule. All the patients in both cohorts recover by day 14 and were in asymptomatic condition and laboratory parameters return to normal values by day 14 after treatment initiation. Adverse events were identified in 31.5% of patients, 28.5% in the control group, and 34.4% in the HeberFERON group, and the most frequent were headaches (17.4%). Conclusions: In a cohort of 63 hospitalized patients between 19 to 82 years-old with positive SARS-CoV-2, HeberFERON significantly negativized the virus on day 4 of treatment when comparing with IFN-alpha2b. Heberon Alpha R also showed efficacy for the treatment of the viral infection. Both treatments were safe and positively impact on the resolution of the symptoms. None of the patients developed severe COVID-19. Key words: COVID-19, treatment, drug, virus negativization, antiviral, interferon combination, SARS CoV-2.