Ayurvedic Herbs Advised for COVID-19 Management: Therapeutic Potential and Clinical Relevance

Coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has spread worldwide. There is no effective medication for COVID-19 as of now, so it would be good to take preventive measures that not only boost our immunity but also fight against infections. The use of traditional Chinese medicine in China to treat COVID-19 patients sets the prototype demonstrating that traditional medicines can contribute to prevention and treatment successfully. In India, the Ministry of AYUSH (Ayurveda, Yoga, Unani, Siddha, Homeop-athy) released a self-care advisory during the COVID-19 crisis as a preventive aspect. This review article discusses the therapeutic potential and clinical relevance of some herbs [(Tulsi (Ocimum sanctum), Haridra (Curcuma longa), Tvaka (Cinnamon), Maricha (Piper longum), Shunthi (Zingi-ber officinale), Munakka (Dried grapes), Lavang (Syzigiumaromaticum), Pudina (Mentha arvensis), and Ajwain (Trachyspermum ammi)] advised by AUYSH to take during COVID-19 infection. They are effective in COVID-19 management, therefore, authors have discussed their detailed traditional uses as therapeutics and spotted scientific insight and clinical significance of the herbs mentioned above along with their mechanistic viewpoint, adequately, on a single platform. Provided information could be a treasure to open up a new research arena on natural products to manage human health crises effectively, caused not only by COVID-19 but also by other infectious diseases.Copyright © 2023 Bentham Science Publishers.

Preparation of the Monoclonal Antibody against the African Swine Fever Virus p54 Protein and Identification of the Antigenic Epitope.

The aim of this paper was to prepare specific monoclonal antibody (mAb) against African swine fever virus (ASFV) p54 protein. The p54 protein was expressed in Escherichia coli expression system and used as the antigen in mAb production. The spleen cells from the immunized BALB/c mice were fused with myeloma cells SP2/0. To screen the positive hybridoma cells, the purified p54 protein was used as envelope antigen for indirect ELISA. After four times' subcloning, the supernatant of hybridoma cells were used to identify mAb subtype, ascites were prepared via in vivo induction method in mice and then the mAb was purified. The titer of the mAb was detected by indirect ELISA, and the specificity of the mAb was identified by cross reactivity assay, IFA and Western blot. According to the predicted secondary structure of p54 protein, using the stepwise truncation method identified the epitope region of mAbs, and labeled the region in tertiary structure of p54 protein. Results were as follows: six hybridoma cells secreting p54 monoclonal antibody were successfully screened and named 28G12-1, 31G7-1, 31G7-2, 35F10-1, 35F10-2, 38D3-1, respectively. The heavy chains of 28G12-1, 31G7-1, and 31G7-2 were IgG2a type, the heavy chains of 35F10-1, 35F10-2, 38D3-1 were IgG1 type, light chains were all kappa chains. The lowest titer of mAb was 1:25 600, and having no cross reaction with PRRSV, PRV, PEDV, PPV, SADS-CoV, PCV2, the specificity was strong. All six monoclonal antibodies could recognize the 127-146 aa on carboxyl end. In this study, ASFV p54 protein and p54 monoclonal antibody were successfully obtained, and the epitopes of six mAbs were identified, these experimental data laid a foundation for the functional research of p54 protein and the study of ASFV epitope vaccine. Copyright © 2023 Editorial Board, Institute of Animal Science of the Chinese Academy of Agricultural Sciences. All rights reserved.

SmRNA PRIME S+N DNA BOOST VACCINATION ELICITS ROBUST IMMUNE RESPONSES in MICE

Background: Currently available COVID-19 vaccination regimens in the US deliver either a homologous spike (S) mRNA prime-boost or a prime-only S DNA adenovirus-vectored antigen to elicit humoral and cell-mediated responses to confer protection against SAR-CoV-2 infection. Alternatively, heterologous vaccination using two different platforms has the potential to enhance and expand immune protection. Addition of a second SARS-CoV-2 antigen, the nucleocapsid (N) protein that is less subject to mutation and elicits vigorous T-cell responses, may also be advantageous. We report immunological responses to homologous and heterologous prime-boost vaccination regimens with a human DNA adenovirus serotype 5 S plus N (AdS+N) and/or a self-amplifying S-only mRNA vaccine (AAAH) delivered with a nanostructured lipid carrier (NLC). Methods: CD-1 mice received homologous or heterologous prime-boost combinations of AdS+N and AAAH. Priming doses were administered on Day 0, booster doses were delivered on Day 21, and mice were euthanized for blood and organ collection on Day 35. Serum was analyzed for anti-S (both wild type and variant) and anti-N IgG subtypes by ELISA. Spleen-resident CD4+ and CD8+ T cells were tested for IFN-γ, TNF-α, and IL-2 production in response to S-WT, S Delta variant and N protein overlapping peptides by intracellular cytokine staining (ICS). Splenocyte cytokine secretion upon stimulation with S-WT/N peptides was also assessed by IFN-γ and IL-4 ELISpot. Serum neutralization of the original Wuhan strain, Delta, and B.1.351 variants was assessed by a pseudovirus neutralization assay. Results: The highest humoral and T-cell responses were seen with the heterologous AAAH prime-AdS+N boost regimen, with a significant increase in T-cell responses relative to homologous vaccination. S protein-binding IgG was similar between wild type and Delta variant S proteins, with a strong/clear Th1/Th2 bias, and T cells responded to S wild type and S Delta peptides with similar levels of cytokine expression. Sera from AAAH prime-AdS+N boost mice showed the ability to neutralize Wuhan D614G, Delta, and B.1.351 (South Africa) variant pseudoviruses at high levels. Conclusion: Heterologous vaccination with the AAAH RNA vaccine prime and an AdS+N DNA boost may provide substantially improved humoral and cell-based immunity against SARS-CoV-2 variants by leveraging the advantages of each vaccine platform technology and by inclusion of immune responses to N.

LONG-TERM IMMUNOGENICITY and EFFECTIVENESS of CD40-TARGETING VACCINATION in COVID-19

Background: Following natural infection or vaccination, the generation of stem cell-like memory T (Tscm) cells is essential for long-term protective immunity to the virus. Tscm cells have the capacity for self-renewal and multipotency. In SARS-CoV2 infection, the emergence of CD8+ Tscm cells is correlated with the number of symptom-free days. The development of a COVID-19 vaccine able to generate CD8+ Tscm cells is of the utmost importance since the emergence of SARS-CoV2 variants of concerns requires maintaining strong and long-lasting immune responses, 2) as an efficient alternative in immunocompromised people who have difficulties raising humoral immune responses. Methods: We have developed a new Dendritic Cell-based vaccine composed of a humanized αCD40 monoclonal antibody fused to the RBD protein in its C-terminal Fc-domains and three T cell epitopes spanning sequences from S and N proteins in its light chains (αCD40-CoV2). Previous studies have shown that this platform elicited durable and robust T-and B-cell responses and is currently in phase I clinical development in HIV. We tested the capacity of two injections of the vaccine (10υg, i.p) given with or without poly(IC) (50υg, i.p) at 3 weeks apart to i) elicit human (hu) B-, and huT-cell responses in NSG mice reconstituted with a Human Immune System (HIS mice), ii) protect against SARS-CoV2 infection in the hCD40xK18hACE2 transgenic mice. Results: We performed AIM assays and intracellular staining on spleen cells of HIS mice stimulated with overlapping peptide pools spanning the sequences of vaccine antigens. We found that both non-adjuvanted and adjuvanted vaccine efficiently induced SARS-CoV2-specific Th1 huCD4+ and huCD8+ T cells in all vaccinees compared to mock animals. SARS-CoV2-specific huCD4+ T cells were polyfunctional. We confirmed the presence of RBD-specific huCD8+ T cells in the vaccinated animals using HLA-I tetramers. A significant proportion of the multimer+ huCD8+ T cells were Tscm (CD45RA+ CD62L+ CD95+) cells in both vaccinated groups. Besides, we detected significant amounts of spike-IgG+ switched huB cells in all vaccinees. In SARS-CoV2 challenge experiments, we further showed that both vaccination settings significantly protected animals with a survival rate of 100%. Conclusion: We demonstrate that the targeting of SARS-CoV-2 epitopes to CD40 induces significant B and T cells with a long-term memory phenotype in HIS mice and the ability of the vaccine to ensure complete protection against SARS-CoV2 infection.

INVESTIGATING CONSERVED SEQUENCE in the SARS-CoV-2 GENOME AS NOVEL VACCINE IMMUNOGEN

Background: It is imperative to investigate novel, broadly conserved coronavirus immunogens as new SARS-CoV-2 variants of concern are continually emerging. The goal of this study was to generate a broadly protective long-term vaccine candidate against potential new variants of SARS-CoV-2 and novel, outbreak coronaviruses. The vaccine immunogen spanned portions of the highly conserved RNA replication machinery (nsp12 and nsp13) (CoV.Con). The vaccine was packaged into a rhesus adenoviral vector (RhAd52.CoV.Con) with the goal of generating robust long-lived CD8+ T-cell responses. Methods: The CoV.Con immunogen was generated by aligning coronavirus sequences to determine the most conserved region. ACE2 carrier and BALB/c mice were immunized intramuscularly with 109 RhAd52.CoV.Con and boosted four weeks later. Splenocytes were harvested four weeks after boost. Cellular immunity was determined through ELIspot and intracellular cytokine stain (ICS). BALB/c mice were primed and boosted with RhAd52.CoV.Con. Four weeks post boost mice were challenged intranasally with mouse adapted SARS-CoV-2. Protection was measured by weight loss and plaque assay. Results: Four weeks post RhAd52.CoV.Con boost immunization, ACE2 carrier and BALB/c mice developed cellular immunity as shown by ELIspot (Fig 1a) and ICS. ACE2 carrier mice cellular immunity showed bias toward nsp12 while BALB/c mice showed nsp13 preference. BALB/c mice were primed and boosted with RhAd52.CoV.Con. Four weeks after boost mice were challenged with mouse adapted SARS-CoV-2. RhAd52.CoV.Con was compared against and combined with a suboptimal dose of RhAd52.S.pp at 4 and 8 weeks post injection. Protection against weight loss (Fig 1b) and viral load (Fig 1c) was minimal although increased RhAd52.S.pp protection was observed from 4 to 8 weeks post immunization. Increased RhAd52.S.pp protection corresponded to increased spike antibody binding and neutralizing titers. Conclusion: Our work investigates a highly conserved coronavirus immunogen, CoV.Con, demonstrating immunogenicity in two mouse strains. While RhAd52. CoV.Con protection in the mouse model was minimal it demonstrates a schema for generating coronavirus immunogens that can protect against multiple different viruses. This work takes the first steps towards generating a long-lived broadly protective T-cell coronavirus vaccine.

Covid-19 vaccines: Efficacy, safety and future prospects

SARS-CoV-2 still remains to be the cause of the Covid-19 pandemic, with more than 200 million infections and over 4.5 million deaths worldwide. With the onset of this pandemic, vaccine development efforts began on an unprecedented scale in the world, and it was seen that some vaccines received global application permissions in a very short period of time. For now, it has been proven that current COVID-19 vaccines are highly effective in protecting against serious illness and death. However, there remains much to learn about the immune response of vaccines and the duration of protection, and how we can further optimize vaccines against new variants. As of today, some inactivated vaccines (eg. Sinovac, Sinopharm, BharatBiotech), mRNA vaccines (eg.Pfizer&Bionthec, Moderna), Non-Replicating Viral Vector ones (eg. Oxford, Astra- Zeneca, Sputnik V) and recombinant protein vaccines (eg.Novovax) have been approved for immediate use, but determining the longterm effectiveness of vaccination with existing vaccines and moving towards adolescent and pediatric applications, as well as the reports on some serious side effects seen especially with mRNA and vector vaccines, remain important issues. In addition, the difficulties of poor countries' access to vaccines, and serious vaccine insecurity or rejection in almost all countries constitute major obstacles against the termination of the pandemic. The extension of the time required for the formation of herd immunity leads to the emergence of new variants. The rapid spread of these variants leading to a significant decrease in the effectiveness of the existing vaccines brings about a need for the modification of the current vaccines. During this period, we developed a different vaccine candidate and two vaccine formulations that can be easily adapted to emerging variants. In our study, a fragment protein (P1;MW: 33 kDa) containing the Receptor-Binding Domain (RBD) in the spike S1 region, a fragment protein in the S2 region (P2;MW: 17.6) and nucleocapsid protein (N;MW: 46 kDa) were expressed in Escherichia coli, and subsequently the recombinant proteins were purified. It was determined that each of these three protein antigens reacted strongly with recovered Covid-19 patient sera. The combination of these three proteins was adsorbed with one adjuvant or two adjuvants led to the development of two formulations. In mouse immunization studies, these vaccine candidates elicited very high titers of anti-P1 IgG and IgG2a, anti-P2 IgG and IgG2a, and anti-N IgG and IgG2a. In the live virus neutralization assays, high virus neutralizing antibody levels were observed, and by obtaining specific interferon-gamma (INF-gamma) from immunized mouse splenocytes, we proved that a good cellular immunity was achieved too. These findings, overall, validated high immunogenicity of the P1 and P2 proteins of the S region and the N protein to order to develop an effective vaccine candidate against SARS CoV-2 infection. In addition, a variant recombinant protein was designed by our group and prepared in the same way. This protein that contains the most important point mutations of the known variants is being intended to be incorporated into our candidate vaccine. Our studies are being conducted in this direction and we aim at starting Phase I trials as soon as possible. In conclusion, such alterations/adaptations in vaccine formulations are highly important to optimize current vaccines. In order to reduce the worldwide effects of SARS-CoV-2 variants, it is necessary to develop new generation COVID-19 vaccines besides an urgent elimination of the vaccine inequality all over the world.

Pharmacologic inhibition of SUMOactivating enzyme potentiates interferon response and T cell-mediated anti-tumor immunity in chronic lymphocytic leukemia (CLL) and lymphoma models

Introduction: CLL is characterized by deficient immunity which clinically manifests as an increased predisposition toward malignancies and infectious complications. T-cells from patients with CLL exhibit a skewed repertoire with a predominance of Tregs as well as impaired immune synapse formation and cytotoxic function. Unlike chemotherapy, novel targeted agents may have beneficial immunomodulatory effects, which may be particularly relevant in the COVID-19 era. Small ubiquitin-like modifier (SUMO) family proteins regulate a variety of cellular processes, including nuclear trafficking, gene transcription, and cell cycle progression, via post-translational modification of target proteins. Sumoylation regulates NFjB signaling, IFN response, and NFAT activation, processes indispensable in immune cell activation. Despite this, the role of sumoylation in T cell biology in the context of cancer is not known. TAK-981 is a small molecule inhibitor of the SUMO-activating enzyme (SAE) that forms a covalent adduct with an activated SUMO protein, thereby preventing its transfer to the SUMO-conjugating enzyme (Ubc9). Here, we investigated the immunomodulatory effects of TAK-981 in CLL. Methods: T cells from patients with CLL were purified using Dynabeads. Activation, proliferation, and apoptosis of CD3+ T cells were studied following T-cell receptor engagement (TCR;aCD3/CD28) with/without 0-1 lM TAK-981. Cytokines were measured after in vitro stimulation. For polarization assays, FACS-sorted naïve CD4+ T cells were cultured for 7 days in control or differentiation media. For gene expression profiling (GEP;Clariom S), RNA was harvested after 3 and 24 h of TCR engagement from FACS-sorted naïve CD4+ T cells. For in vivo immunization experiments, CD4+KJ1-26+ cells were inoculated IV into BALB/cJ mice. Mice received 100 mg IV ovalbumin ± R848 followed by TAK-981 7.5 mg/kg or vehicle control IV twice weekly for 10 days before spleen collection. Both recipient and transplanted splenocytes were analyzed. For analysis of tumor-infiltrating lymphocytes (TILs), BALB/c mice were injected with 1×106 A20 lymphoma cells and treated as above. TAK-981 was provided by Millennium Pharmaceuticals, Inc. (Cambridge, MA, USA). Results: T cells from patients with CLL demonstrated high baseline protein sumoylation that slightly increased following TCR engagement. Treatment with TAK-981 significantly downregulated SUMO1 and SUMO2/3-modified protein levels, yet did not disrupt early TCR signaling as evidenced by sustained ZAP70, p65/NFjB, and NFAT activation detected by immunoblotting, immunocytochemistry, and GEP. Treatment with TAK-981 resulted in dose-dependent upregulation of the early activation marker CD69 in CD4+ T cells following 72 and 96 h of TCR stimulation vs. control. Meanwhile, the expression of CD25, HLA-DR, and CD40L was delayed in the presence of TAK-981. Interestingly, CD38, an IFN response target, was induced 2-fold in TAK-981-treated cells after 24 h and persisted at high levels at subsequent timepoints. T cell proliferation was reduced in the presence of high (1 lM) but not low/intermediate concentrations of TAK-981, accompanied by reduced S phase entry and decreased synthesis of IL- 2. However, T cells did not undergo apoptosis under those conditions. Targeting SAE in either control or Th1/Treg polarizing conditions facilitated an increase in IFNc and loss of FoxP3 expression (accompanied by decreased IL-2/STAT5), suggesting a shift toward Th1 and away from Treg phenotype, respectively. GEP (Reactome, GSEA) confirmed a dramatically upregulated IFN response in TAK-981-treated CD4+ naïve T cells. Furthermore, targeting SAE enhanced degranulation (CD107a), IFNc, and perforin secretion in cytotoxic CD8+ T cells and potentiated T cell cytotoxicity in allogeneic assays with lymphoma cells (OCI-LY3, U2932) as targets. Consistent with our in vitro data, OVA-stimulated transplanted transgenic KJ1-26+ splenocytes, as well as total CD4+ T cells from recipient mice treated with TAK-981 in vivo exhibited a significant reduction in express on of FoxP3 and an increased production of IFNc. In the A20 syngeneic model, treatment with TAK-981 similarly downregulated FoxP3 expression in CD4+ TILs and induced IFNc secretion in CD8+ TILs. Conclusion: Using a combination of in vitro and in vivo experiments, we demonstrate that pharmacologic targeting of sumoylation with TAK-981 does not impair proximal TCR signaling in T cells obtained from patients with CLL, but leads to rebalancing toward healthy immune T cell subsets via induction of IFN response and downmodulation of Tregs. These data provide a strong rationale for continued investigation of TAK-981 in CLL and lymphoid malignancies.