Potential Therapeutic Targets and Vaccine Development for SARS-CoV-2/COVID-19 Pandemic Management: A Review on the Recent Update.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is a highly pathogenic novel virus that has caused a massive pandemic called coronavirus disease 2019 (COVID-19) worldwide. Wuhan, a city in China became the epicenter of the outbreak of COVID-19 in December 2019. The disease was declared a pandemic globally by the World Health Organization (WHO) on 11 March 2020. SARS-CoV-2 is a beta CoV of the Coronaviridae family which usually causes respiratory symptoms that resemble common cold. Multiple countries have experienced multiple waves of the disease and scientific experts are consistently working to find answers to several unresolved questions, with the aim to find the most suitable ways to contain the virus. Furthermore, potential therapeutic strategies and vaccine development for COVID-19 management are also considered. Currently, substantial efforts have been made to develop successful and safe treatments and SARS-CoV-2 vaccines. Some vaccines, such as inactivated vaccines, nucleic acid-based, and vector-based vaccines, have entered phase 3 clinical trials. Additionally, diverse small molecule drugs, peptides and antibodies are being developed to treat COVID-19. We present here an overview of the virus interaction with the host and environment and anti-CoV therapeutic strategies; including vaccines and other methodologies, designed for prophylaxis and treatment of SARS-CoV-2 infection with the hope that this integrative analysis could help develop novel therapeutic approaches against COVID-19.

Principles of Allergen Immunotherapy and Its Clinical Application in China: Contrasts and Comparisons with the USA.

Allergen immunotherapy (AIT) for allergic rhinitis (AR), asthma, and other allergic diseases has developed quickly. House dust mite (HDM), Artemisia (wormwood), Humulus japonicus (Japanese hop), Alternaria alternata, and Cladosporium herbarum are the five most common inhalant allergens in China. AIT has been performed in China for over 60 years. With the support of the Chinese Medical Association (CMA) and the Chinese Medical Doctors Association (CMDA), the Chinese College of Allergy and Asthma (CCAA) was established in 2016 as a specialized branch of CDMA and is the main certification authority for AIT. Chinese allergists and scientists have made tremendous progress in the development of AIT. There have been many publications by Chinese allergists and scientists worldwide encompassing original research studies, systematic reviews, case studies, and clinical trials. Currently, conventional subcutaneous immunotherapy (SCIT) is the preferred AIT in China, but sublingual immunotherapy (SLIT) is beginning to gain recognition. An increasing number of clinical trials have been conducted to investigate the clinical efficacy and side effects of SLIT and SCIT. In China, HDM is the only commercial standardized allergen extracts in clinical use, whereas the others are crude allergen extracts. Besides standardized allergen extracts, other forms of hypoallergenic extracts are still being investigated and developed in China. Immunotherapy in China is similar to that in the USA in which allergen extracts can be mixed for SCIT. However, allergen extracts cannot be mixed for SCIT in Europe.

Cancer DNA vaccines: current preclinical and clinical developments and future perspectives.

The recent developments in immuno-oncology have opened an unprecedented avenue for the emergence of vaccine strategies. Therapeutic DNA cancer vaccines are now considered a very promising strategy to activate the immune system against cancer. In the past, several clinical trials using plasmid DNA vaccines demonstrated a good safety profile and the activation of a broad and specific immune response. However, these vaccines often demonstrated only modest therapeutic effects in clinical trials due to the immunosuppressive mechanisms developed by the tumor. To enhance the vaccine-induced immune response and the treatment efficacy, DNA vaccines could be improved by using two different strategies. The first is to increase their immunogenicity by selecting and optimizing the best antigen(s) to be inserted into the plasmid DNA. The second strategy is to combine DNA vaccines with other complementary therapies that could improve their activity by attenuating immunosuppression in the tumor microenvironment or by increasing the activity/number of immune cells. A growing number of preclinical and clinical studies are adopting these two strategies to better exploit the potential of DNA vaccination. In this review, we analyze the last 5-year preclinical studies and 10-year clinical trials using plasmid DNA vaccines for cancer therapy. We also investigate the strategies that are being developed to overcome the limitations in cancer DNA vaccination, revisiting the rationale for different combinations of therapy and the different possibilities in antigen choice. Finally, we highlight the most promising developments and critical points that need to be addressed to move towards the approval of therapeutic cancer DNA vaccines as part of the standard of cancer care in the future.

Recombinant Listeria promotes tumor rejection by CD8+ T cell-dependent remodeling of the tumor microenvironment.

Agents that remodel the tumor microenvironment (TME), prime functional tumor-specific T cells, and block inhibitory signaling pathways are essential components of effective immunotherapy. We are evaluating live-attenuated, double-deleted Listeria monocytogenes expressing tumor antigens (LADD-Ag) in the clinic. Here we show in numerous mouse models that while treatment with nonrecombinant LADD induced some changes in the TME, no antitumor efficacy was observed, even when combined with immune checkpoint blockade. In contrast, LADD-Ag promoted tumor rejection by priming tumor-specific KLRG1+PD1loCD62L- CD8+ T cells. These IFNγ-producing effector CD8+ T cells infiltrated the tumor and converted the tumor from an immunosuppressive to an inflamed microenvironment that was characterized by a decrease in regulatory T cells (Treg) levels, a proinflammatory cytokine milieu, and the shift of M2 macrophages to an inducible nitric oxide synthase (iNOS)+CD206- M1 phenotype. Remarkably, these LADD-Ag-induced tumor-specific T cells persisted for more than 2 months after primary tumor challenge and rapidly controlled secondary tumor challenge. Our results indicate that the striking antitumor efficacy observed in mice with LADD-based immunotherapy stems from TME remodeling which is a direct consequence of eliciting potent, systemic tumor-specific CD8+ T cells.

Advances & challenges in leptospiral vaccine development.

Considerable progress has been made in the field of leptospiral vaccines development since its first use as a killed vaccine in guinea pigs. Despite the fact that the immunity conferred is restricted to serovars with closely related lipopolysaccharide antigen, certain vaccines have remained useful, especially in endemic regions, for the protection of high-risk individuals. Other conventional vaccines such as the live-attenuated vaccine and lipopolysaccharide (LPS) vaccine have not gained popularity due to the reactive response that follows their administration and the lack of understanding of the pathogenesis of leptospirosis. With the recent breakthrough and availability of complete genome sequences of Leptospira, development of novel vaccine including recombinant protein vaccine using reverse vaccinology approaches has yielded encouraging results. However, factors hindering the development of effective leptospiral vaccines include variation in serovar distribution from region to region, establishment of renal carrier status following vaccination and determination of the dose and endpoint titres acceptable as definitive indicators of protective immunity. In this review, advancements and progress made in LPS-based vaccines, killed- and live-attenuated vaccines, recombinant peptide vaccines and DNA vaccines against leptospirosis are highlighted.

A nano silicon adjuvant enhances inactivated transmissible gastroenteritis vaccine through activation the Toll-like receptors and promotes humoral and cellular immune responses.

Inactivated transmissible gastroenteritis virus (TGEV) vaccines are widely used in swine herds in China. These are limited, however, by the need to elicit both humoral and cellular immunity, as well as the efficiency of adjuvants. In this study, a 70-nm nano silicon particle was applied with inactivated TGEV vaccine in mice, and its immune-enhancing effects and mechanism of action investigated. We found that nano silicon applied with inactivated TGEV vaccine induced high antibody titers, increase IL-6, TNF-α and IFN-γ expression, and stimulate CD3+ T cell proliferation with a high CD4+/CD8+ T lymphocyte ratio. Nano silicon could quickly activate innate and adaptive immunity by stimulating Toll-like receptor signaling pathways, indicating that the nano silicon adjuvant enhanced long-term humoral and early cellular immune responses when combined with inactivated TGEV vaccine. Nano silicon could be considered for use as an antigen- carrier and adjuvant for veterinary vaccines.

Preclinical study and clinical trial of a novel therapeutic vaccine against multi-drug resistant tuberculosis.

[Purpose] Multi-drug resistant (MDR), Mycobacterium tuberculosis (TB) is a big problem in the world. We have developed novel TB therapeutic vaccine (HVJ-E/HSP65 DNA +IL-12 DNA). [Methods and Results] DNA vaccine expressing TB heat shock protein 65 and IL-12 was delivered by the hemagglutinating virus of Japan (HVJ)-envelope. This vaccine provided remarkable protective efficacy and strong therapeutic efficacy against MDR-TB and XDR-TB in murine models. Furthermore, this vaccine provided therapeutic efficacy of prolongation of survival time of TB infected monkeys and augmented the immune responses. Therefore, the preclinical tests were studied for clinical trial. The injection of 100 µg of the vaccine /mouse i.m. three times in two weeks induced significantly strong production of IFN-γ and IL-2. 100 µg and 200 µg DNA vaccine/mouse i.m. augmented the production of these cytokines compared with 25 µg DNA vaccine/mouse i.m.. The ratio of 100 µg pDNA to 1AU HVJ-E enhanced the production of IFN-γ and IL-2. The decrease in the number of M. tuberculosis in liver of mice was observed by the vaccination of 100µg pDNA. By using these conditions, safety pharmacology study and toxicology test is being studied in monkeys administered by GMP level DNA vaccines. By the toxicology test using monkeys, high dose GMP level vaccine/ monkey is administrated. Safety pharmacological study of repeated administration is also being investigated in GLP level. Furthermore, we have planned to do clinical phase I trial. Targets are human patients with MDR-TB. The safety and tolerability of the vaccine will be evaluated. [Conclusion and recommendations] These data indicate that our novel vaccine might be useful against tuberculosis including XDR-TB and MDR-TB for human therapeutic clinical applications.

Bacterial toxin's DNA vaccine serves as a strategy for the treatment of cancer, infectious and autoimmune diseases.

DNA vaccination -a third generation vaccine-is a modern approach to stimulate humoral and cellular responses against different diseases such as infectious diseases, cancer and autoimmunity. These vaccines are composed of a gene that encodes sequences of a desired protein under control of a proper (eukaryotic or viral) promoter. Immune response following DNA vaccination is influenced by the route and the dose of injection. In addition, antigen presentation following DNA administration has three different mechanisms including antigen presentation by transfected myocytes, transfection of professional antigen presenting cells (APCs) and cross priming. Recently, it has been shown that bacterial toxins and their components can stimulate and enhance immune responses in experimental models. A study demonstrated that DNA fusion vaccine encoding the first domain (DOM) of the Fragment C (FrC) of tetanus neurotoxin (CTN) coupled with tumor antigen sequences is highly immunogenic against colon carcinoma. DNA toxin vaccines against infectious and autoimmune diseases are less studied until now. All in all, this novel approach has shown encouraging results in animal models, but it has to go through adequate clinical trials to ensure its effectiveness in human. However, it has been proven that these vaccines are safe, multifaceted and simple and can be used widely in organisms which may be of advantage to public health in the near future. This paper outlines the mechanism of the action of DNA vaccines and their possible application for targeting infectious diseases, cancer and autoimmunity.

Long-Term Reduction of High Blood Pressure by Angiotensin II DNA Vaccine in Spontaneously Hypertensive Rats.

Recent research on vaccination has extended its scope from infectious diseases to chronic diseases, including Alzheimer disease, dyslipidemia, and hypertension. The aim of this study was to design DNA vaccines for high blood pressure and eventually develop human vaccine therapy to treat hypertension. Plasmid vector encoding hepatitis B core-angiotensin II (Ang II) fusion protein was injected into spontaneously hypertensive rats using needleless injection system. Anti-Ang II antibody was successfully produced in hepatitis B core-Ang II group, and antibody response against Ang II was sustained for at least 6 months. Systolic blood pressure was consistently lower in hepatitis B core-Ang II group after immunization, whereas blood pressure reduction was continued for at least 6 months. Perivascular fibrosis in heart tissue was also significantly decreased in hepatitis B core-Ang II group. Survival rate was significantly improved in hepatitis B core-Ang II group. This study demonstrated that Ang II DNA vaccine to spontaneously hypertensive rats significantly lowered high blood pressure for at least 6 months. In addition, Ang II DNA vaccines induced an adequate humoral immune response while avoiding the activation of self-reactive T cells, assessed by ELISPOT assay. Future development of DNA vaccine to treat hypertension may provide a new therapeutic option to treat hypertension.

Immunotherapeutic approaches in prostate cancer: combinations and clinical integration.

Despite multiple immunologic approaches with peptide, protein, and DNA vaccines, no single therapy has induced complete remission or maintained durability of response in patients with castration-resistant prostate cancer (CRPC). Historically, immunotherapy has had limited effect on solid tumors with the exception of melanoma and renal cell carcinomas, which have been deemed as immunologic cancers given their potential for remissions either spontaneously or after removal of the primary lesion. There is considerable excitement about using an immunotherapy in combination with biologic agents such as checkpoint inhibitors, cytokines, other vaccines, or chemotherapy. Sipuleucel-T represents one of several novel immunologic therapeutic approaches to treat prostate cancer in addition to other solid tumors. It is the first in its class of autologous cellular therapies to demonstrate safety and an overall survival benefit in patients with asymptomatic or minimally symptomatic CRPC and represents a unique treatment method that may be further enhanced with other agents. Although sipuleucel-T can be used as a foundation on which to build and enhance future immunologic clinical trials, other exciting strategies are in development that may be easily integrated into the algorithm of current care.

Preclinical safety and tolerability of a repeatedly administered human leishmaniasis DNA vaccine.

The leishmaniases are a complex of vector-borne diseases caused by protozoan parasites of the genus Leishmania. LEISHDNAVAX is a multi-antigen, T-cell epitope-enriched DNA vaccine candidate against human leishmaniasis. The vaccine candidate has been proven immunogenic and showed prophylactic efficacy in preclinical studies. Here, we describe the safety testing of LEISHDNAVAX in naive mice and rats, complemented by the demonstration of tolerability in Leishmania-infected mice. Biodistribution and persistence were examined following single and repeated intradermal (i.d.) administration to rats. DNA vectors were distributed systemically but did not accumulate upon repeated injections. Although vector DNA was cleared from most other tissues within 60 days after the last injection, it persisted in skin at the site of injection and in draining lymph nodes. Evaluation of single-dose and repeated-dose toxicity of the vaccine candidate after i.d. administration to naive, non-infected mice did not reveal any safety concerns. LEISHDNAVAX was also well tolerated in Leishmania-infected mice. Taken together, our results substantiate a favorable safety profile of LEISHDNAVAX in both naive and infected animals and thus, support the initiation of clinical trials for both preventive and therapeutic applications of the vaccine.

Safety and immunogenicity of therapeutic DNA vaccine with antiviral drug in chronic HBV patients and its immunogenicity in mice.

BACKGROUND & AIMS: Here, we evaluated the safety and immunogenicity of hepatitis B virus (HBV) DNA vaccine, HB-110, in mice and Korean patients with chronic hepatitis B (CHB) undergoing adefovir dipivoxil (ADV) treatment. METHODS: For animal study, mice (BALB/c or HBV transgenic) were immunized with mHB-110, and T-cell and antibody responses were evaluated. For clinical study, 27 patients randomly received either ADV alone or ADV in combination with HB-110. Liver function tests, serum HBV DNA levels and the presence of HBeAg/anti-HBe were analysed. T-cell responses were estimated by ELISPOT and FACS analysis. RESULTS: mHB-110 induced higher T-cell and antibody responses than mHB-100 in mice. No adverse effects were observed by HB-110 cotreated with ADV. HBV-specific T-cell responses were induced in a portion of patients in medium to high dose of HB-110. Interestingly, HB-110 exhibited positive effects on ALT normalization and maintenance of HBeAg seroconversion. One patient, who received high dose of HB-110 exhibited HBeAg seroconversion during vaccination, which correlated with vaccine-induced T-cell responses without ALT elevation. CONCLUSIONS: HB-110 was safe and tolerable in CHB patients. In contrast to results in animal models, HB-110 in Korean patients exhibited weaker capability of inducing HBV-specific T-cell responses and HBeAg seroconversion than HB-100 in Caucasian patients. As Asian patients, who are generally infected via vertical transmission, appeared to have higher level of immune tolerance than Caucasian, novel approaches for breaking immune tolerance rather than enhancing immunogenicity may be more urgently demanded to develop effective therapeutic HBV DNA vaccines.

Chemo-immunotherapy using saffron and its ingredients followed by E7-NT (gp96) DNA vaccine generates different anti-tumor effects against tumors expressing the E7 protein of human papillomavirus.

Saffron and its components have been suggested as promising candidates for cancer prevention. Carotenoids and monoterpene aldehydes are two potent ingredients of saffron. The goal of the current study was to investigate the anti-tumor effect of chemo-immunotherapy using saffron and its ingredients followed by E7-NT (gp96) DNA vaccine against tumors expressing the E7 protein of human papillomavirus. The in vitro cytotoxic and apoptotic effects of aqueous saffron extract and its components were evaluated in malignant TC-1 and non-malignant COS-7 cell lines. Then, multimodality treatments using E7-NT (gp96) DNA vaccine combined with saffron extract and its ingredients as well as single-modality treatments were tested for their efficacy in inhibiting large and bulky tumor growth. Saffron and its components exerted a considerable anti-tumor effect through prevention of cell growth and stimulation of programmed cell death. Furthermore, 100 % of mice treated with crocin were tumor-free, in contrast to DNA vaccine alone (~66.7 %) and DNA + crocin (~33.3 %) indicating the high potency of crocin as a chemotherapeutic agent. Interestingly, the multimodality treatment using DNA vaccine along with picrocrocin augmented the anti-tumor effects of picrocrocin. Thus, the combination of DNA vaccine with saffron extract and crocin at certain concentrations did not potentiate protective and therapeutic effects compared to mono-therapies for the control of TC-1 tumors.

Emerging therapies for food allergy.

Food allergy is a common condition for which there are currently no approved treatments except avoidance of the allergenic food and treatment of accidental reactions. There are several potential treatments that are under active investigation in animal and human studies, but it is not yet clear what the best approach may be. Here, we review approaches that are currently in clinical trials, including oral, sublingual, and epicutaneous immunotherapy, immunotherapy combined with anti-IgE, and Chinese herbal medicine as well as approaches that are in preclinical or early clinical investigation, including modified protein immunotherapy, adjuvants, DNA vaccines, and helminth administration. We discuss the importance of fully exploring the risks and benefits of any treatment before it is taken to general clinical practice and the need for clarity about the goals of treatment.

Can anthocyanins improve maintenance therapy of Ph(+) acute lymphoblastic leukaemia?

Chemotherapy and tyrosine kinase inhibitors provide high remission rates. However, prognosis of adult patients with Philadelphia chromosome-positive acute lymphoblastic leukaemia (Ph(+) ALL) still remains poor. Because most adults eventually relapse without allogeneic stem cell transplantation, which is not available for all patients, novel strategies are required for relapse prevention. As the integrity of the immune system is essential for the control of remaining leukaemia cells, we compared the efficacy of anthocyanins, imatinib and a DNA-based vaccine as non-immunosuppressant components with 6-mercaptopurine (6-MP) to control minimal residual disease in vitro and in vivo using different leukaemia cell lines and syngeneic mice. Proliferation of Ph(+) ALL was significantly better inhibited by anthocyanin-rich berry extract or imatinib compared with 6-MP. Although anthocyanins induced apoptosis in some leukaemia cell lines, the level of caspase-3, caspase-8 and caspase-9 was significantly lower compared with imatinib and 6-MP. When used as single components, anthocyanins and imatinib mesylate failed to eradicate pre-existing Ph(+) ALL in syngeneic mice, while 6-MP led to 10% and DNA vaccination to 56% survival. Intriguingly, only the combination of DNA vaccination with berry extract but not with the isolated anthocyanin, cyanidin-3-rutinoside or imatinib further increased leukaemia-free and overall survival, and 90% of lethally challenged mice survived. We suggest that induction and enhancement of a leukaemia-specific immunity by DNA vaccination and anthocyanin-rich berry extract can also decrease the relapse rate in patients with Ph(+) ALL. Furthermore, this approach may serve as strategy for maintenance therapy of other malignancies.

A novel adjuvant Ling Zhi-8 enhances the efficacy of DNA cancer vaccine by activating dendritic cells.

DNA vaccine has been suggested to use in cancer therapy, but the efficacy remains to be improved. The immunostimulatory effect of a fungal immunomodulatory protein Ling Zhi-8 (LZ-8) isolated from Ganoderma lucidum has been reported. In this study, we tested the adjuvanticity of LZ-8 for HER-2/neu DNA vaccine against p185(neu) expressing tumor MBT-2 in mice. We found that recombinant LZ-8 stimulated mouse bone marrow-derived dendritic cells (DCs) via TLR4 and its stimulatory effect was not due to any microbe contaminant. In addition, LZ-8 enhanced the ability of DCs to induce antigen-specific T cell activation in vitro and in a subunit vaccine model in vivo. Surprisingly, LZ-8 cotreatment strongly improved the therapeutic effect of DNA vaccine against MBT-2 tumor in mice. This increase in antitumor activity was attributed to the enhancement of vaccine-induced Th1 and CTL responses. Consistent with the results from DCs, the promoting effect of LZ-8 on DNA vaccine was diminished when the MBT-2 tumor cells were grown in TLR4 mutant mice. Thus, we concluded that LZ-8 may be a promising adjuvant to enhance the efficacy of DNA vaccine by activating DCs via TLR4.

Drug delivery trends in clinical trials and translational medicine: challenges and opportunities in the delivery of nucleic acid-based therapeutics.

The ability to deliver nucleic acids (e.g., plasmid DNA, antisense oligonucleotides, siRNA) offers the potential to develop potent vaccines and novel therapeutics. However, nucleic acid-based therapeutics are still in their early stages as a new category of biologics. The efficacy of nucleic acids requires that these molecules be delivered to the interior of the target cell, which greatly complicates delivery strategies and compromises efficiency. Due to the safety concerns of viral vectors, synthetic vectors such as liposomes and polymers are preferred for the delivery of nucleic acid-based therapeutics. Yet, delivery efficiencies of synthetic vectors in the clinic are still too low to obtain therapeutic levels of gene expression. In this review, we focus on some key issues in the field of nucleic acid delivery such as PEGylation, encapsulation and targeted delivery and provide some perspectives for consideration in the development of improved synthetic vectors.

Fighting mycobacterial infections by antibiotics, phytochemicals and vaccines.

Buruli ulcer is a neglected disease caused by Mycobacterium ulcerans and represents the world's third most common mycobacterial infection. It produces the polyketide toxins, mycolactones A, B, C and D, which induce apoptosis and necrosis. Clinical symptoms are subcutaneous nodules, papules, plaques and ulcerating oedemae, which can enlarge and destroy nerves and blood vessels and even invade bones by lymphatic or haematogenous spread (osteomyelitis). Patients usually do not suffer from pain or systematic inflammation. Surgery is the treatment of choice, although recurrence is common and wide surgical excisions including healthy tissues result in significant morbidity. Antibiotic therapy with rifamycins, aminoglycosides, macrolides and quinolones also improves cure rates. Still less exploited treatment options are phytochemicals from medicinal plants used in affected countries. Vaccination against Buruli ulcer is still in its infancy.

BHT-3009, a myelin basic protein-encoding plasmid for the treatment of multiple sclerosis.

Even though the etiology of multiple sclerosis (MS) remains largely unknown, research data support the hypothesis that autoimmunity plays a major role in disease development. Several disease-modifying agents have been approved for the treatment of MS; however, there is still a need for antigen-specific treatments that combine efficacy and safety. DNA vaccination represents a new therapeutic alternative in this respect. Preclinical studies in different models of autoimmunity have demonstrated that injection of plasmid DNA encoding a self-antigen in mice restores self-tolerance, leaving immunity against infectious and tumor antigens intact. Based on this evidence, the first DNA vaccine for MS has been created. Bayhill Therapeutic Inc's BHT-3009 encodes full-length, human myelin basic protein (MBP), and has recently been evaluated in a phase I/II and a phase II clinical trial. BHT-3009 was safe and well tolerated in both trials, inducing immune tolerance that extended beyond MBP to other myelin antigens. In addition, a reduction in the number of active lesions was observed, which was accompanied by a decrease in clinical relapse rates, particularly in patients with high immunological activity at baseline. BHT-3009 appears to be a promising new approach for the treatment of MS, although further clinical trials are warranted to confirm the early findings.