Identification of a novel inhibitor of SARS-CoV-2 main protease: an in silico, biochemical, and cell-based approach.

The recurrent nature of coronavirus outbreaks, severity of the COVID-19 pandemic, rapid emergence of novel variants, and concerns over the effectiveness of existing vaccines against novel variants have highlighted the need to develop therapeutic interventions. Targeted efforts to identify inhibitors of crucial viral proteins are the preferred strategy. In this study, we screened FDA-approved and natural product libraries using in silico approach for potential hits against the SARS-CoV-2 main protease (Mpro) and experimentally validated their potency using in vitro biochemical and cell-based assays. Seven potential hits were identified through in silico screening and were subsequently evaluated in SARS-CoV-2-based cell-free assays, followed by testing in the HCoV-229E-based culture system. Of the tested compounds, 4-(3,4-dihydroxyphenyl)-6,7-dihydroxy-1-isopropyl-1H-benzofuro[3,2-b]pyrazolo[4,3-e]pyridin-3(2H)-one (PubChem CID:71755304, hereafter referred to as STL522228) exhibited significant antiviral activity. Subsequently, its potential as a novel COVID therapeutic molecule was validated in the SARS-CoV-2-culture system, where STL522228 demonstrated superior antiviral activity (EC50 = 0.44 µm) compared to Remdesivir (EC50 = 0.62 µm). Based on these findings, we report the strong anti-coronavirus activity of STL522228, and propose that it as a promising pan-coronavirus Mpro inhibitor for further experimental and preclinical validation.

Adenoviruses as gene/vaccine delivery vectors: promises and pitfalls.

INTRODUCTION: Adenoviruses (Ads) have attracted researchers from across the disciplines for several reasons. Their ability to recombine with and express the heterologous genes encouraged their development as gene/vaccine delivery vectors. Preclinical and clinical trials using these vectors have shown significant promise for their future development while highlighting certain limitations. AREAS COVERED: Ad vector development, clinical application of these vectors in gene therapy and vaccine development, and promises that these vectors offered for future development are discussed. Also, various factors affecting their in vivo efficacy as well as the strategies being pursued to overcome these obstacles are described. EXPERT OPINION: Decades of efforts have resulted in understanding various aspects of human Ad 5 biology as well as the in vivo behavior of vectors derived from this serotype. While this vector has shown great potential, some of the limitations highlighted in recent studies have reinforced the quest for an ideal vector derived from novel Ads. Combinatorial approaches to address the various limitations of the existing vectors must be pursued to develop novel vectors with enhanced clinical potential.

Transplacental rotavirus IgG interferes with immune response to live oral rotavirus vaccine ORV-116E in Indian infants.

The lower immune response and efficacy of live oral rotavirus (RV) vaccines tested in developing countries may be due in part to high levels of pre-existing RV antibodies transferred to the infant from mother via the placenta. The candidate RV vaccine strain 116E was isolated from a newborn indicating that it might grow well even in the presence of this transplacental rotavirus antibody. Since the immune response to this vaccine among infants in the Indian subcontinent has been greater than that of the commercially licensed vaccines, we questioned whether this might be due to the ability of RV 116E to grow well in infants despite the presence of maternal RV antibody. To this end, we tested pre-immunization sera from Indian infants enrolled in a phase Ia/IIb trial of candidate RV vaccine ORV-116E for transplacental RV IgG to see whether it affected the immune responses and seroconversion to the vaccine. We found that the high titers of transplacental RV IgG diminished the immune responses of infants to ORV-116E vaccine. However, the vaccine was able to overcome the inhibitory effect of this RV IgG in a dose-dependent manner. This report clearly demonstrates the interference of maternal antibody on RV vaccine immunogenicity in infants in a field study as well as the ability of ORV-116E to overcome this interference when used at a higher dose.

Clinical development of IMOJEV ®--a recombinant Japanese encephalitis chimeric vaccine (JE-CV).

INTRODUCTION: Japanese encephalitis (JE) is a disease of the central nervous system (CNS) caused by Japanese encephalitis virus (JEV). JE is endemic in most of the South-East Asian countries and in some parts of the Western Pacific. As mosquito control is ineffective, currently vaccination is the only available control measure. A mouse brain-derived inactivated JE vaccine (MBDV) has been in use for a long time; however, it is not feasible for mass vaccination due to the ethical and safety issues. With the World Health Organization (WHO) appealing for the development of novel, safe and affordable JE vaccines, several vaccine candidates have been developed in the recent times and IMOJEV ® is one among them. AREAS COVERED: This review presents a brief account of various developmental, immunological and ethical issues related to IMOJEV® and provides an in-depth account of its clinical development and efficacy in comparison to other JE vaccines. EXPERT OPINION: IMOJEV® is a safe and efficacious vaccine. If made affordable through financial assistance from health agencies or by its production in set ups where operational costs are lower, it may become an ideal vaccine for mass vaccination in JE endemic regions.

IMOJEV(®): a Yellow fever virus-based novel Japanese encephalitis vaccine.

Japanese encephalitis (JE) is a disease of the CNS caused by Japanese encephalitis virus (JEV). The disease appears in the form of frequent outbreaks in most south- and southeast Asian countries and the virus has become endemic in several areas. There is no licensed therapy available and disease control by vaccination is considered to be most effective. Mouse brain-derived inactivated JE vaccines, although immunogenic, have several limitations in terms of safety, availability and requirement for multiple doses. Owing to these drawbacks, the WHO called for the development of novel, safe and more efficacious JE vaccines. Several candidate vaccines have been developed and at least three of them that demonstrated strong immunogenicity after one or two doses of the vaccine in animal models were subsequently tested in various clinical trials. One of these vaccines, IMOJEV(®) (JE-CV and previously known as ChimeriVax™-JE), is a novel recombinant chimeric virus vaccine, developed using the Yellow fever virus (YFV) vaccine vector YFV17D, by replacing the cDNA encoding the envelope proteins of YFV with that of an attenuated JEV strain SA14-14-2. IMOJEV was found to be safe, highly immunogenic and capable of inducing long-lasting immunity in both preclinical and clinical trials. Moreover, a single dose of IMOJEV was sufficient to induce protective immunity, which was similar to that induced in adults by three doses of JE-VAX(®), a mouse brain-derived inactivated JE vaccine. Recently, Phase III trials evaluating the immunogenicity and safety of the chimeric virus vaccine have been successfully completed in some JE-endemic countries and the vaccine manufacturers have filed an application for vaccine registration. IMOJEV may thus be licensed for use in humans as an improved alternative to the currently licensed JE vaccines.

Expression of Japanese encephalitis virus envelope protein in transgenic tobacco plants.

The virus envelope (E) protein of Japanese encephalitis virus induces virus-neutralizing antibodies and is therefore a potential vaccine antigen. In a mammalian system, co-expression of another viral structural protein prM is necessary for proper expression and folding of E protein. Transgenic tobacco plants were produced carrying JEV cDNA encoding prM and E proteins under the control of the CaMV 35S promoter. E protein, however, was not detectable in these plants. In vitro translation studies showed that the presence of the prM sequence inhibited transgene expression in the plant system. Accordingly, JEV E protein could be expressed in transgenic tobacco plants only without the prM protein.

A dose-escalation safety and immunogenicity study of live attenuated oral rotavirus vaccine 116E in infants: a randomized, double-blind, placebo-controlled trial.

BACKGROUND: Rotavirus infections cause approximately 122,000 deaths among Indian children annually. METHODS: The neonatal rotavirus candidate vaccine 116E was tested in a double-blind, placebo-controlled dose-escalation trial in India. Two doses of the Vero cell-adapted vaccine were evaluated. One hundred eighty-seven infants received a vaccine dose of 1 X 10(4) focus-forming units (ffu) and 182 received a dose of 1 X 10(5) ffu in a 1:1 randomization with placebo recipients. Infants received the vaccine at 8, 12, and 16 weeks, separately from routine vaccines. RESULTS: No significant differences in clinical adverse events or laboratory toxicity were observed between vaccine and placebo recipients. There were no vaccine-related serious adverse events. A 4-fold increase in rotavirus immunoglobulin A titer was observed in 66.7% and 64.5% of infants after the first administration and in 62.1% and 89.7% of infants after 3 administrations of doses of 1 X 10(4) ffu and 1 X 10(5) ffu, respectively; the differences between these groups and placebo recipients were statistically significant. CONCLUSIONS: Three administrations of vaccine doses of 1 X 10(4) ffu and 1 X 10(5) ffu were safe. The 1 X 10(5)-ffu dose of 116E demonstrated a robust immune response after 3 administrations. These favorable results warrant further development of the vaccine candidate and provide optimism that vaccinating infants in the developing world will prevent serious sequelae of rotavirus infection. Clinical trials registration. NCT00439660 and ISRCTN57452882 .

Seroprevalence of neutralizing antibodies to adenovirus type 5 among children in India: implications for recombinant adenovirus-based vaccines.

We determined the levels of adenovirus 5 (Ad5) neutralizing antibodies in children in India less than 2 years of age. The results clearly show an age-dependent increase in Ad5-specific immunity, with 7- to 12-month-old children having the lowest levels of Ad5 immunity. This opens up the scope for the use of recombinant Ad5-based vaccines in this age group.

DNAzyme-mediated inhibition of Japanese encephalitis virus replication in mouse brain.

Japanese encephalitis virus (JEV) is an arthropod-borne flavivirus with a single-stranded RNA genome containing non-coding regions (NCRs) at its 5' and 3'-ends. The NCRs have flavivirus-conserved sequences that are important for virus replication. Here we describe DNAzymes (Dzs) that cleave the RNA sequence of the 3'-NCR of JEV genome in vitro. The nuclease-resistant Dzs, containing phosphorothioate linkages, were efficiently taken up by mouse neuronal and glial cells, and addition of a continuous stretch of 10 guanosine residues (poly-(G)(10)) to the 3'-end of a Dz led to its enhanced delivery to cells containing scavenger receptors (ScRs). These novel Dzs inhibited JEV replication in cultured mouse cells of neuronal and macrophage origin. JEV is a neurotropic virus that actively replicates in mouse brain. Here we show that intra-cerebral (i.c.) administration of a poly-(G)(10)-tethered, phosphorothioated Dz in JEV-infected mice led to more than 99.99% inhibition of virus replication in brain, resulting in a dose-dependent extended lifespan or complete recovery of the infected animals. This is the first report of in vivo application of a Dz to control a virus infection in an animal model.

Immunization with recombinant adenovirus synthesizing the secretory form of Japanese encephalitis virus envelope protein protects adenovirus-exposed mice against lethal encephalitis.

Replication-defective recombinant adenoviruses (RAds) were constructed that synthesized the pre-membrane and envelope (E) proteins of Japanese encephalitis virus (JEV). Recombinant virus RAdEa synthesized Ea, the membrane-anchored E protein, and RAdEs synthesized Es, the secretory E protein. Compared with RAdEs, RAdEa replicated poorly in HEK 293A cells and synthesized lower amounts of E protein. Oral immunization of mice with RAds generated low titers of anti-JEV antibodies that had little JEV neutralizing activity. Intra-muscular (IM) immunization of mice with either RAd generated high titers of anti-JEV antibodies. Interestingly, RAdEa induced only low titers of JEV neutralizing antibodies. Titers were significantly higher in case of RAdEs immunization. Splenocytes from mice immunized IM with RAds secreted large amounts of interferon-gamma and moderate amounts of interleukin-5 in the presence of JEV and showed cytotoxic activity against JEV-infected cells. Naïve mice immunized IM with RAdEs showed complete protection against a lethal dose of JEV given intra-cerebrally. In order to study the effect of the pre-existing adenovirus 5 (Ad5) immunity on the outcome of the RAdEs immunization, mice were exposed to Ad5 through IM or intra-nasal (IN) routes before immunization with RAdEs. Mice exposed to Ad5 through the IN route, when immunized with RAdEs given IM, or those exposed to Ad5 through the IM route, when immunized with RAdEs given IN, were completely protected against lethal JEV challenge.

Effect of cytokine-encoding plasmid delivery on immune response to Japanese encephalitis virus DNA vaccine in mice.

We have previously shown that immunization of mice with plasmid pMEa synthesizing Japanese encephalitis virus (JEV) envelope protein induced anti-JEV humoral and cellular immune responses. We now show that intra-muscular co-administration of mice with pMEa and pGM-CSF, encoding murine granulocyte-macrophage colony-stimulating factor or pIL-2, encoding murine interleukin-2 given 4 days after pMEa, augmented anti-JEV antibody titers. This did not enhance the level of protection in immunized mice against JEV. However, intra-dermal co-administration of pMEa and pGM-CSF in mice using the gene gun, enhanced anti-JEV antibody titers resulting in an increased level of protection in mice against lethal JEV challenge.

Oral immunization of mice with Japanese encephalitis virus envelope protein synthesized in Escherichia coli induces anti-viral antibodies.

In order to evaluate the possibility of developing an oral vaccine against Japanese encephalitis virus (JEV), mice were fed with recombinant JEV envelope (E) protein synthesized in Escherichia coli. The protein was administered orally to mice with or without an immunostimulatory cytosine-phosphate-guanosine (CpG) motif containing synthetic oligodeoxynucleotide (ODN) as an adjuvant. The immunized mice made high-titered anti-E and anti-JEV antibodies. Mice immunized with JEV E protein along with the ODN adjuvant produced higher antibody titers and these were predominantly IgG2a type. These antibodies, however, failed to neutralize JEV activity in vitro, and the immunization did not protect the mice against lethal JEV challenge. Splenocytes from the immunized mice secreted large amounts of interferon (IFN)-gamma and showed proliferation in the presence of JEV E protein. Our results indicate that JEV E protein delivered orally to mice together with ODN generated both humoral and cellular immune responses to JEV, and these were of the Th1 type.

Japanese encephalitis virus (JEV) is an important cause of encephalitis among children in Cuddalore district, Tamil Nadu, India.

BACKGROUND: Japanese encephalitis (JE) is endemic in Cuddalore district, Tamil Nadu (TN), Southern India. The reports of JE cases from the local hospitals did not reflect the actual disease burden. It is likely that these cases were attending the nearby referral hospitals, for want of better treatment facilities. OBJECTIVES: Between July 2002 and February 2003, a pilot study was undertaken to examine whether JE was a component of paediatric acute encephalitis syndrome (AES) reported to two major referral hospitals adjacent to Cuddalore, and to map the distribution of the JE cases. STUDY DESIGN: A total of 58 hospitalized children [0-15 years] with AES were investigated. Other than the routine laboratory investigations, either CSF or sera or both [depending on the availability] collected from these children were analyzed at Center for Research in Medical Entomology, Madurai (TN) for JEV-antigen, antibody detection, virus isolation and virus genome detection by indirect immunofluorescence, MAC enzyme linked immunosorbent assay (ELISA), insect bioassay and by reverse transcriptase polymerase chain reaction (RT-PCR), respectively. RESULTS: JE was established in 17 (29%) of 58 AES cases; half of the AES cases [31/58, 53%] and 59% [10/17] of JE cases were confined to JE-endemic areas in Cuddalore district. The JE confirmation scored by different assays varied according to the clinical phase of the illness. The attack rate was high among the children aged 3-8 years. The monthly distribution of acute encephalitic syndrome cases followed the distribution of JE cases [coinciding with the rainy season in this region] suggesting encephalitis of JE origin. CONCLUSION: In JE-endemic areas, the actual JE burden can be estimated by the collection of JE case reports from the local hospitals and from the referral hospitals. Building of diagnostic facilities in hospitals for JE is necessary to achieve this goal.

Immunogenicity and protective efficacy in mice of a formaldehyde-inactivated Indian strain of Japanese encephalitis virus grown in Vero cells.

P20778, an Indian strain of Japanese encephalitis virus (JEV) obtained from Vellore in the Southern India, was grown in Vero cells cultured on microcarriers in a spinner flask. The virus was formalin-inactivated and its immunogenicity and protective efficacy in mice were tested in comparison with a commercially available vaccine. Our studies indicated that formalin-inactivated JEV P20778 induced high levels of protective immunity in mice. Virus inactivation with formalin at 22 degrees C, which required shorter incubation period, was found to be as good or better to virus inactivation at 4 degrees C for generating high titers of anti-JEV antibodies. Similarly, the 22 degrees C-inactivated virus generated JEV neutralizing antibody titers as good or higher than those induced by the 4 degrees C-inactivated virus. Thus, for the vaccine production, inactivation of JEV with formalin at 22 degrees C would be a preferred method as it is faster and does not require cold room storage.