Publications by authors named "Navin Khanna"

78 Publications

Double-Antigen Lateral Flow Immunoassay for the Detection of Anti-HIV-1 and -2 Antibodies Using Upconverting Nanoparticle Reporters.

Sensors (Basel) 2021 Jan 6;21(2). Epub 2021 Jan 6.

Department of Biotechnology, University of Turku, 20520 Turku, Finland.

Rapid diagnostic tests (RDTs) are often used for the detection of anti-human immunodeficiency virus (HIV) antibodies in remote locations in low- and middle-income countries (LMIC) with low or limited access to central laboratories. The typical format of an RDT is a lateral flow assay (LFA) with visual interpretation prone to subjectivity. This risk of misinterpretation can be overcome with luminescent upconverting nanoparticle reporters (UCNPs) measured with a miniaturized easy-to-use reader instrument. An LFA with UCNPs for anti-HIV-1/2 antibodies was developed and the assay performance was evaluated extensively with challenging patient sample panels. Sensitivity ( = 145) of the UCNP-LFA was 96.6% (95% CI: 92.1-98.8%) and specificity ( = 309) was 98.7% (95% CI: 96.7-99.7%). Another set of samples ( = 200) was used for a comparison between the UCNP-LFA and a conventional visual RDT. In this comparison, the sensitivities for HIV-1 were 96.4% (95% CI: 89.8-99.3%) and 97.6% (95% CI: 91.6-99.7%), for the UCNP-LFA and conventional RDT, respectively. The specificity was 100% (95% CI: 96.4-100%) for both assays. The developed UCNP-LFA demonstrates the applicability of UCNPs for the detection of anti-HIV antibodies. The signal measurement is done by a reader instrument, which may facilitate automated result interpretation, archiving and transfer of data from de-centralized locations.
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http://dx.doi.org/10.3390/s21020330DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7825344PMC
January 2021

Upconverting nanoparticle reporter-based highly sensitive rapid lateral flow immunoassay for hepatitis B virus surface antigen.

Anal Bioanal Chem 2021 Feb 23;413(4):967-978. Epub 2020 Nov 23.

Translational Health Science and Technology Institute, NCR Biotech Science Cluster, Faridabad, 121001, India.

Detection of hepatitis B Virus surface antigen (HBsAg) is an established method for diagnosing both acute and chronic hepatitis B virus (HBV) infection. In addition to enzyme immunoassays (EIAs), rapid diagnostic tests (RDTs) are available for the detection of HBsAg in resource-poor settings. However, the available RDTs have inadequate sensitivity and therefore are not suitable for diagnosis of patients with low levels of HBsAg and for blood screening. To provide a high-sensitivity RDT, we developed a lateral flow immunoassay (LFIA) for HBsAg utilizing upconverting nanoparticle (UCNP) reporter. The UCNP-LFIA can use whole blood, serum, or plasma and the results can be read in 30 min using a reader device. When compared with a commercial conventional visually read LFIA, the developed UCNP-LFIA had a Limit of Detection (LoD) of 0.1 IU HBsAg/ml in spiked serum, whereas the LoD of the conventional LFIA was 3.2 IU HBsAg/ml. The developed UCNP-LFIA fulfills the WHO criterion for blood screening (LoD ≤ 0.13 IU HBsAg/ml) in terms of LoD. The UCNP-LFIA and conventional LFIA were evaluated with well-characterized sample panels. The UCNP-LFIA detected 20/24 HBsAg-positive samples within the HBsAg Performance Panel and 8/10 samples within the Mixed Titer Performance Panel, whereas the conventional LFIA detected 8/24 and 4/10 samples in these panels, respectively. The performance of the assays was further evaluated with HBsAg-positive (n = 108) and HBsAg-negative (n = 315) patient samples. In comparison with a central laboratory test, UCNP-LFIA showed 95.4% (95% CI: 89.5-98.5%) sensitivity whereas sensitivity of the conventional LFIA was 87.7% (95%CI: 79.9-93.3%).
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http://dx.doi.org/10.1007/s00216-020-03055-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7813740PMC
February 2021

Ultrasensitive and Robust Point-of-Care Immunoassay for the Detection of Malaria.

Anal Chem 2020 12 23;92(24):15766-15772. Epub 2020 Nov 23.

Translational Health Science and Technology Institute, NCR Biotech Science Cluster, 3rd Milestone, Faridabad-Gurgaon Expressway, Faridabad 121001, Haryana, India.

malaria is widespread in the tropical and subtropical regions of the world. There is ongoing effort to eliminate malaria from endemic regions, and sensitive point-of-care (POC) diagnostic tests are required to support this effort. However, current POC tests are not sufficiently sensitive to detect in asymptomatic individuals. After extensive optimization, we have developed a highly sensitive and robust POC test for the detection of infection. The test is based on upconverting nanophosphor-based lateral flow (UCNP-LF) immunoassay. The developed UCNP-LF test was validated using whole blood reference panels containing samples at different parasite densities covering eight strains of from different geographical areas. The limit of detection was compared to a WHO-prequalified rapid diagnostic test (RDT). The UCNP-LF achieved a detection limit of 0.2-2 parasites/μL, depending on the strain, which is 50- to 250-fold improvement in analytical sensitivity over the conventional RDTs. The developed UCNP-LF is highly stable even at 40 °C for at least 5 months. The extensively optimized UCNP-LF assay is as simple as the conventional malaria RDTs and requires 5 μL of whole blood as sample. Results can be read after 20 min from sample addition, with a simple photoluminescence reader. In the absence of a reader device at the testing site, the strips after running the test can be transported and read at a central location with access to a reader. We have found that the test and control line signals are stable for at least 10 months after running the test. The UCNP-LF has potential for diagnostic testing of both symptomatic and asymptomatic individuals.
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http://dx.doi.org/10.1021/acs.analchem.0c02748DOI Listing
December 2020

Antibody-Dependent Enhancement: A Challenge for Developing a Safe Dengue Vaccine.

Front Cell Infect Microbiol 2020 22;10:572681. Epub 2020 Oct 22.

Translational Health Group, Molecular Medicine Division, International Centre for Genetic Engineering and Biotechnology, New Delhi, India.

In 2019, the United States Food and Drug Administration accorded restricted approval to Sanofi Pasteur's Dengvaxia, a live attenuated vaccine (LAV) for dengue fever, a mosquito-borne viral disease, caused by four antigenically distinct dengue virus serotypes (DENV 1-4). The reason for this limited approval is the concern that this vaccine sensitized some of the dengue-naïve recipients to severe dengue fever. Recent knowledge about the nature of the immune response elicited by DENV viruses suggests that all LAVs have inherent capacity to predominantly elicit antibodies (Abs) against the pre-membrane (prM) and fusion loop epitope (FLE) of DENV. These antibodies are generally cross-reactive among DENV serotypes carrying a higher risk of promoting Antibody-Dependent Enhancement (ADE). ADE is a phenomenon in which suboptimal neutralizing or non-neutralizing cross-reactive antibodies bind to virus and facilitate Fcγ receptor mediated enhanced entry into host cells, followed by its replication, and thus increasing the cellular viral load. On the other hand, antibody responses directed against the host-cell receptor binding domain of DENV envelope domain-III (EDIII), exhibit a higher degree of type-specificity with lower potential of ADE. The challenges associated with whole DENV-based vaccine strategies necessitate re-focusing our attention toward the designed dengue vaccine candidates, capable of inducing predominantly type-specific immune responses. If the designed vaccines elicited predominantly EDIII-directed serotype specific antibodies in the absence of prM and FLE antibodies, this could avoid the ADE phenomenon largely associated with the prM and FLE antibodies. The generation of type-specific antibodies to each of the four DENV serotypes by the designed vaccines could avoid the immune evasion mechanisms of DENVs. For the enhanced vaccine safety, all dengue vaccine candidates should be assessed for the extent of type-specific (minimal ADE) vs. cross-reactive (ADE promoting) neutralizing antibodies. The type-specific EDIII antibodies may be more directly related to protection from disease in the absence of ADE promoted by the cross-reactive antibodies.
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http://dx.doi.org/10.3389/fcimb.2020.572681DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7642463PMC
October 2020

Dengue and Zika virus infections are enhanced by live attenuated dengue vaccine but not by recombinant DSV4 vaccine candidate in mouse models.

EBioMedicine 2020 Oct 16;60:102991. Epub 2020 Sep 16.

Translational Health Group, Molecular Medicine Division, International Centre for Genetic Engineering & Biotechnology, New Delhi, India. Electronic address:

Background: A tetravalent live attenuated dengue vaccine, Dengvaxia, sensitised naïve recipients to severe dengue illness upon a subsequent natural dengue infection and is suspected to be due to antibody-dependent enhancement (ADE). ADE has also been implicated in the severe neurological outcomes of Zika virus (ZIKV) infection. It has become evident that cross-reactive antibodies targeting the viral pre-membrane protein and fusion-loop epitope are ADE-competent. A pre-clinical tetravalent dengue sub-unit vaccine candidate, DSV4, eliminates these ADE-competent epitopes.

Methods: We compared protective efficacy and ADE-competence of murine polyclonal antibodies induced by DSV4, Dengvaxia and an 'in house' tetravalent mixture of all four laboratory DENV strains, TV DENV, using established mouse models.

Findings: DSV4-induced antibodies, known to be predominantly type-specific, provided significant protection against lethal DENV challenge, but did not promote ADE of either DENV or ZIKV infection in vivo. Antibodies elicited by Dengvaxia and TV DENV, which are predominantly cross-reactive, not only failed to offer protection against lethal DENV challenge, but also promoted ADE of both DENV and ZIKV infection in vivo.

Interpretation: Protective efficacy against DENV infection may be linked to the induction of neutralising antibodies which are type-specific rather than cross-reactive. Whole virus-based dengue vaccines may be associated with ADE risk, despite their potent virus-neutralising capacity. Vaccines designed to eliminate ADE-competent epitopes may help eliminate/minimise ADE risk.

Funding: This study was supported partly by ICGEB, India, the National Biopharma Mission, DBT, Government of India, Sun Pharmaceutical Industries Limited, India, and NIAID, NIH, USA.
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http://dx.doi.org/10.1016/j.ebiom.2020.102991DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7501058PMC
October 2020

Enhanced cell density cultivation and rapid expression-screening of recombinant Pichia pastoris clones in microscale.

Sci Rep 2020 05 4;10(1):7458. Epub 2020 May 4.

Translational Health Science and Technology Institute, NCR Biotech Science Cluster, Faridabad, Haryana, India.

Cultivation of yeast Pichia pastoris in the microtiter plate, for optimisation of culture conditions, and expression screening of transformants has gained significance in recent years. However, in the microtiter plate, it has been challenging to attain cell densities similar to well-aerated shake-flask culture, due to the poor mixing resulting in oxygen limitation. To solve this problem, we investigated the influence of multiple cultivation parameters on P. pastoris cell growth, including the architecture of 96-deepwell plate (96-DWP), shaking throw diameter, shaking frequency, culture volume/well, and media composition. In the optimised conditions, a cell density of OD ~50 (dry cell weight ~13 g/L) with >99% cell viability was achieved in the casamino acids supplemented buffered-minimal-media in 300 to 1000 μl culture volume/well. We have devised a simplified method for coating of the culture supernatant on the polystyrene surface for immunoassay. Clones for secretory expression of envelope domain III of dengue virus serotype-1 under the control of inducible and constitutive promoter were screened using the developed method. Described microscale cultivation strategy can be used for rapid high-throughput screening of P. pastoris clones, media optimization, and high-throughput recombinant protein production. The knowledge gained through this work may also be applied, to other suspension cultures, with some modifications.
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http://dx.doi.org/10.1038/s41598-020-63995-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7198582PMC
May 2020

Tetravalent Dengue Vaccine in Healthy Children.

N Engl J Med 2020 04;382(18):1769-1770

International Centre for Genetic Engineering and Biotechnology, New Delhi, India

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http://dx.doi.org/10.1056/NEJMc2000987DOI Listing
April 2020

Zika virus envelope nanoparticle antibodies protect mice without risk of disease enhancement.

EBioMedicine 2020 Apr;54:102738

Recombinant Gene Products Group, Molecular Medicine Division, International Centre for Genetic Engineering & Biotechnology, Aruna Asaf Ali Marg, New Delhi 110067, India; Translational Health Science & Technology Institute, NCR Biotech Science Cluster, Faridabad, India. Electronic address:

Background: Zika virus (ZIKV), an arbovirus capable of causing neurological abnormalities, is a recognised human pathogen, for which a vaccine is required. As ZIKV antibodies can mediate antibody-dependent enhancement (ADE) of dengue virus (DENV) infection, a ZIKV vaccine must not only protect against ZIKV but must also not sensitise vaccinees to severe dengue.

Methods: The N-terminal 80% of ZIKV envelope protein (80E) was expressed in Pichia pastoris and its capacity to self-assemble into particulate structures evaluated using dynamic light scattering and electron microscopy. Antigenic integrity of the 80E protein was evaluated using ZIKV-specific monoclonal antibodies. Its immunogenicity and protective efficacy were assessed in BALB/c and C57BL/6 Stat2 mice, respectively. Its capacity to enhance DENV and ZIKV infection was assessed in AG129 and C57BL/6 Stat2 mice, respectively.

Findings: ZIKV-80E protein self-assembled into discrete nanoparticles (NPs), which preserved the antigenic integrity of neutralising epitopes on E domain III (EDIII) and elicited potent ZIKV-neutralising antibodies predominantly against this domain in BALB/c mice. These antibodies conferred statistically significant protection in vivo (p = 0.01, Mantel-Cox test), and did not exacerbate sub-lethal DENV-2 or ZIKV challenges in vivo.

Interpretation: Yeast-expressed ZIKV-80E, which forms highly immunogenic EDIII-displaying NPs, elicits ZIKV EDIII-specific antibodies capable of offering significant protection in vivo, without the potential risk of ADE upon subsequent DENV-2 or ZIKV infection. This offers a promising vaccine candidate for further development.

Funding: This study was supported partly by ICGEB, India, and by NIAID, USA.
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http://dx.doi.org/10.1016/j.ebiom.2020.102738DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7186774PMC
April 2020

Pichia pastoris-expressed Zika virus envelope domain III on a virus-like particle platform: design, production and immunological evaluation.

Pathog Dis 2019 04;77(3)

Recombinant Gene Products Group, Molecular Medicine Division, International Centre for Genetic Engineering & Biotechnology, Aruna Asaf Ali Marg, New Delhi -110067, India.

Zika virus (ZIKV) is an arbovirus which shares antigenic similarity and the mosquito vector with dengue viruses (DENVs). ZIKV is a neurotropic virus capable of causing congenital neurodevelopmental birth defects. As ZIKV antibodies (Abs) can potentially enhance infection by DENVs, a preventive ZIKV vaccine must be designed to eliminate antibody dependent enhancement of infection. We developed a Zika Subunit Vaccine (ZSV) consisting of two proteins, ZS and S, in a genetically pre-determined ratio of 1:4, using the methylotrophic yeast Pichia pastoris. ZS is an in-frame fusion of ZIKV envelope domain III with the Hepatitis B virus (HBV) surface antigen, and S is the un-fused HBV surface antigen. Using specific monoclonal Abs we showed the presence of ZS and S in the co-purified material which were found to co-assemble into virus-like particles (VLPs), based on dynamic light scattering and electron microscopic analyses. These VLPs were immunogenic in BALB/c mice, eliciting Abs capable of neutralizing ZIKV reporter virus particles. Further, the VLP-induced Abs did not enhance a sub-lethal DENV-2 challenge in AG129 mice. This important safety feature, coupled to the well-documented advantage of P. pastoris expression system, warrants further exploration of ZSV VLP as a possible vaccine candidate.
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http://dx.doi.org/10.1093/femspd/ftz026DOI Listing
April 2019

Dengue vaccine development: Global and Indian scenarios.

Int J Infect Dis 2019 Jul 23;84S:S80-S86. Epub 2019 Jan 23.

Recombinant Gene Products Group, Molecular Medicine Division, International Centre for Genetic Engineering and Biotechnology, Aruna Asaf Ali Marg, New Delhi, India; Translational Health Science and Technology Institute, NCR Biotech Science Cluster, Faridabad, India. Electronic address:

India is home to nearly a third of the global population at risk of dengue, a viral disease caused by four antigenically and genetically distinct dengue viruses. Clinical illness following dengue virus infection can either be mild and self-limiting dengue fever or severe dengue hemorrhagic fever/dengue shock syndrome, with potentially fatal consequences. A live attenuated vaccine known as Dengvaxia, developed by Sanofi, was licensed in 2015. Following this, long-term follow-up of the Sanofi phase III efficacy trial participants has revealed potential safety concerns. This vaccine, which appears to predispose dengue-naïve recipients to an increased risk of hospitalization in the future, is recommended by the World Health Organization only for adults with a history of prior dengue virus infection. A safe and efficacious dengue vaccine continues to be sought globally. India has joined these efforts in recent years, and is poised to initiate the clinical development of two candidates in the near future, one licensed from abroad and the other developed indigenously. This article provides a glimpse of India's efforts to develop dengue vaccines in the context of the global dengue vaccine development and evaluation landscape and highlights key issues and questions confronting the dengue vaccine community.
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http://dx.doi.org/10.1016/j.ijid.2019.01.029DOI Listing
July 2019

Next generation designer virus-like particle vaccines for dengue.

Expert Rev Vaccines 2019 02 17;18(2):105-117. Epub 2019 Jan 17.

a Recombinant Gene Products Group, Molecular Medicine Division , International Centre for Genetic Engineering & Biotechnology , New Delhi , India.

Introduction: A safe and efficacious vaccine for dengue continues to be an unmet public health need. The recent licensing of a dengue vaccine (Dengvaxia) developed by Sanofi has brought to the fore the safety issue of vaccine-induced infection enhancement.

Areas Covered: This article focuses on two new yeast-produced tetravalent dengue envelope domain III-displaying virus-like particulate vaccine candidates reported in early 2018 and reviews the rationale underlying their design, and pre-clinical data which suggest that these may offer promising alternate options.

Expert Commentary: These are the only vaccine candidates so far to have demonstrated the induction of primarily serotype-specific neutralizing antibodies to all dengue virus serotypes in experimental animals. Interestingly, these antibodies lack infection-enhancing potential when evaluated using the AG129 mouse model.
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http://dx.doi.org/10.1080/14760584.2019.1562909DOI Listing
February 2019

Serodiagnostic evaluation of recombinant CdtB of S. Typhi as a potential candidate for acute typhoid.

Immunol Res 2018 08;66(4):503-512

Centre for Bio-design & Diagnostics, Translational Health Science and Technology Institute, Faridabad, Haryana, India.

Typhoid fever caused by human restricted Salmonella typhi presents a considerable health burden on developing South-Asian nations like India. The suboptimal sensitivity and specificity associated with culture-based isolation of etiological agent and the extensively used surface antigen-based serological assays often lead to misdiagnosis and inappropriate antimicrobial treatment. The increasing reports of the emergence of resistant strains and undefined disease burden signify the critical need for an inexpensive, reliable, easy-to-use, and highly sensitive diagnostic test for typhoid fever. Utilizing S. typhi-specific and immunogenic antigens in sero-diagnostic assays could lead to precise diagnosis of acute typhoid and prompt treatment. In this study, we report cloning, expression, and purification of recombinant Cytolethal distending toxin subunit B (CdtB) of S. typhi, which is reported to be highly specific, immunogenic, and expressed only upon S. typhi infection. We further evaluated the purified recombinant CdtB for its diagnostic potential in an IgM-based indirect ELISA format using 33 human samples. Twenty-one serum samples from blood culture confirmed cases (n = 21) of typhoid and 12 samples from healthy controls (n = 12) were tested. The assay showed sensitivity of 100% and specificity of 83.3% respectively with positive and negative predictive values of 91.3 and 100% respectively. Efficient detection of specific IgM antibodies indicates that CdtB could be highly valuable in sero-diagnosis of acute typhoid and rapid screening of clinical samples.
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http://dx.doi.org/10.1007/s12026-018-9009-4DOI Listing
August 2018

Dengue envelope-based 'four-in-one' virus-like particles produced using Pichia pastoris induce enhancement-lacking, domain III-directed tetravalent neutralising antibodies in mice.

Sci Rep 2018 06 5;8(1):8643. Epub 2018 Jun 5.

Recombinant Gene Products Group, Molecular Medicine Division, International Centre for Genetic Engineering & Biotechnology, New Delhi, India.

Dengue is a significant public health problem worldwide, caused by four antigenically distinct mosquito-borne dengue virus (DENV) serotypes. Antibodies to any given DENV serotype which can afford protection against that serotype tend to enhance infection by other DENV serotypes, by a phenomenon termed antibody-dependent enhancement (ADE). Antibodies to the viral pre-membrane (prM) protein have been implicated in ADE. We show that co-expression of the envelope protein of all four DENV serotypes, in the yeast Pichia pastoris, leads to their co-assembly, in the absence of prM, into tetravalent mosaic VLPs (T-mVLPs), which retain the serotype-specific antigenic integrity and immunogenicity of all four types of their monomeric precursors. Following a three-dose immunisation schedule, the T-mVLPs elicited EDIII-directed antibodies in mice which could neutralise all four DENV serotypes. Importantly, anti-T-mVLP antibodies did not augment sub-lethal DENV-2 infection of dengue-sensitive AG129 mice, based on multiple parameters. The 'four-in-one' tetravalent T-mVLPs possess multiple desirable features which may potentially contribute to safety (non-viral, prM-lacking and ADE potential-lacking), immunogenicity (induction of virus-neutralising antibodies), and low cost (single tetravalent immunogen produced using P. pastoris, an expression system known for its high productivity using simple inexpensive media). These results strongly warrant further exploration of this vaccine candidate.
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http://dx.doi.org/10.1038/s41598-018-26904-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5988708PMC
June 2018

A quinoline compound inhibits the replication of dengue virus serotypes 1-4 in Vero cells.

Antivir Ther 2018 ;23(5):385-394

Department of Biological Sciences, Birla Institute of Technology & Science Pilani, Hyderabad Campus, Hyderabad, India.

Background: The global occurrence of dengue, a mosquito-​borne viral disease caused by four distinct dengue viruses (DENV-1, -2, -3 and -4), is reported to have increased approximately 30-fold in the last 50 years, causing approximately 400 million infections a year. A limited use, sub-optimal live attenuated dengue vaccine has become available recently. It is becoming apparent that antibodies to DENVs can promote infection by Zika virus (ZIKV), a related mosquito-borne flavivirus. A drug to treat these flaviviral infections continues to be an unmet public health need.

Methods: We screened an 'in-house' library of approximately 2,000 small molecules for inhibitors of cloned DENV-2 protease. Putative inhibitor binding to DENV-2 protease was analysed by in silico docking. Anti-DENV activity was analysed by monitoring viral antigen synthesis by ELISA, viral RNA synthesis by reverse-transcription​ coupled to real-time polymerase chain reaction and infectious virus production by plaque assay, in DENV-infected Vero cells.

Results: A quinoline derivative, BT24, was identified for the first time as a potent inhibitor of the cloned DENV-2 protease (half maximal inhibitory concentration [IC]=0.5 µM). In silico analysis revealed that BT24 binds to an allosteric site in the vicinity of the active site of DENV-2 protease. Cell-based assays demonstrated that BT24 can inhibit all four DENVs in infected Vero cells.

Conclusions: BT24 is a DENV-2 protease inhibitor which manifests the capacity to inhibit the replication of all four DENVs in cultured cells. It may provide a lead for a pan-DENV inhibitory drug.
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http://dx.doi.org/10.3851/IMP3231DOI Listing
September 2019

-Expressed Bivalent Virus-Like Particulate Vaccine Induces Domain III-Focused Bivalent Neutralizing Antibodies without Antibody-Dependent Enhancement .

Front Microbiol 2017 9;8:2644. Epub 2018 Jan 9.

Recombinant Gene Products Group, Molecular Medicine Division, International Centre for Genetic Engineering and Biotechnology, New Delhi, India.

Dengue, a significant public health problem in several countries around the world, is caused by four different serotypes of mosquito-borne dengue viruses (DENV-1, -2, -3, and -4). Antibodies to any one DENV serotype which can protect against homotypic re-infection, do not offer heterotypic cross-protection. In fact, cross-reactive antibodies may augment heterotypic DENV infection through antibody-dependent enhancement (ADE). A recently launched live attenuated vaccine (LAV) for dengue, which consists of a mixture of four chimeric yellow-fever/dengue vaccine viruses, may be linked to the induction of disease-enhancing antibodies. This is likely related to viral interference among the replicating viral strains, resulting in an unbalanced immune response, as well as to the fact that the LAV encodes prM, a DENV protein documented to elicit ADE-mediating antibodies. This makes it imperative to explore the feasibility of alternate ADE risk-free vaccine candidates. Our quest for a non-replicating vaccine centered on the DENV envelope (E) protein which mediates virus entry into the host cell and serves as an important target of the immune response. Serotype-specific neutralizing epitopes and the host receptor recognition function map to E domain III (EDIII). Recently, we found that -expressed DENV E protein, of all four serotypes, self-assembled into virus-like particles (VLPs) in the absence of prM. Significantly, these VLPs displayed EDIII and elicited EDIII-focused DENV-neutralizing antibodies in mice. We now report the creation and characterization of a novel non-replicating recombinant particulate vaccine candidate, produced by co-expressing the E proteins of DENV-1 and DENV-2 in . The two E proteins co-assembled into bivalent mosaic VLPs (mVLPs) designated as mE1E2 VLPs. The mVLP, which preserved the serotype-specific antigenic integrity of its two component proteins, elicited predominantly EDIII-focused homotypic virus-neutralizing antibodies in BALB/c mice, demonstrating its efficacy. In an ADE model, mE1E2 VLP-induced antibodies lacked discernible ADE potential, compared to the cross-reactive monoclonal antibody 4G2, as evidenced by significant reduction in the levels of IL-6 and TNF-α, suggesting inherent safety. The results obtained with these bivalent mVLPs suggest the feasibility of incorporating the E proteins of DENV-3 and DENV-4 to create a tetravalent mVLP vaccine.
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http://dx.doi.org/10.3389/fmicb.2017.02644DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5768101PMC
January 2018

A tetravalent virus-like particle vaccine designed to display domain III of dengue envelope proteins induces multi-serotype neutralizing antibodies in mice and macaques which confer protection against antibody dependent enhancement in AG129 mice.

PLoS Negl Trop Dis 2018 01 8;12(1):e0006191. Epub 2018 Jan 8.

Recombinant Gene Products Group, Molecular Medicine Division, International Centre for Genetic Engineering and Biotechnology, New Delhi, India.

Background: Dengue is one of the fastest spreading vector-borne diseases, caused by four antigenically distinct dengue viruses (DENVs). Antibodies against DENVs are responsible for both protection as well as pathogenesis. A vaccine that is safe for and efficacious in all people irrespective of their age and domicile is still an unmet need. It is becoming increasingly apparent that vaccine design must eliminate epitopes implicated in the induction of infection-enhancing antibodies.

Methodology/principal Findings: We report a Pichia pastoris-expressed dengue immunogen, DSV4, based on DENV envelope protein domain III (EDIII), which contains well-characterized serotype-specific and cross-reactive epitopes. In natural infection, <10% of the total neutralizing antibody response is EDIII-directed. Yet, this is a functionally relevant domain which interacts with the host cell surface receptor. DSV4 was designed by in-frame fusion of EDIII of all four DENV serotypes and hepatitis B surface (S) antigen and co-expressed with unfused S antigen to form mosaic virus-like particles (VLPs). These VLPs displayed EDIIIs of all four DENV serotypes based on probing with a battery of serotype-specific anti-EDIII monoclonal antibodies. The DSV4 VLPs were highly immunogenic, inducing potent and durable neutralizing antibodies against all four DENV serotypes encompassing multiple genotypes, in mice and macaques. DSV4-induced murine antibodies suppressed viremia in AG129 mice and conferred protection against lethal DENV-4 virus challenge. Further, neither murine nor macaque anti-DSV4 antibodies promoted mortality or inflammatory cytokine production when passively transferred and tested in an in vivo dengue disease enhancement model of AG129 mice.

Conclusions/significance: Directing the immune response to a non-immunodominant but functionally relevant serotype-specific dengue epitope of the four DENV serotypes, displayed on a VLP platform, can help minimize the risk of inducing disease-enhancing antibodies while eliciting effective tetravalent seroconversion. DSV4 has a significant potential to emerge as a safe, efficacious and inexpensive subunit dengue vaccine candidate.
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http://dx.doi.org/10.1371/journal.pntd.0006191DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5774828PMC
January 2018

Evaluation of a pan-serotype point-of-care rapid diagnostic assay for accurate detection of acute dengue infection.

Diagn Microbiol Infect Dis 2017 Mar 30;87(3):229-234. Epub 2016 Sep 30.

Division of Infectious Diseases, St. John's Research Institute, St. John's National Academy of Health Sciences, Bangalore, 560034, India; International Vaccine Access Center, Johns Hopkins Bloomberg School of Public Health, Baltimore, USA. Electronic address:

The catastrophic rise in dengue infections in India and globally has created a need for an accurate, validated low-cost rapid diagnostic test (RDT) for dengue. We prospectively evaluated the diagnostic performance of NS1/IgM RDT (dengue day 1) using 211 samples from a pediatric dengue cohort representing all 4 serotypes in southern India. The dengue-positive panel consisted of 179 dengue real-time polymerase chain reaction (RT-PCR) positive samples from symptomatic children. The dengue-negative panel consisted of 32 samples from dengue-negative febrile children and asymptomatic individuals that were negative for dengue RT-PCR/NS1 enzyme-linked immunosorbent assay/IgM/IgG. NS1/IgM RDT sensitivity was 89.4% and specificity was 93.8%. The NS1/IgM RDT showed high sensitivity throughout the acute phase of illness, in primary and secondary infections, in different severity groups, and detected all 4 dengue serotypes, including coinfections. This NS1/IgM RDT is a useful point-of-care assay for rapid and reliable diagnosis of acute dengue and an excellent surveillance tool in our battle against dengue.
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http://dx.doi.org/10.1016/j.diagmicrobio.2016.09.020DOI Listing
March 2017

Recombinant Dengue Virus 4 Envelope Glycoprotein Virus-Like Particles Derived from Pichia pastoris are Capable of Eliciting Homotypic Domain III-Directed Neutralizing Antibodies.

Am J Trop Med Hyg 2017 Jan 7;96(1):126-134. Epub 2016 Nov 7.

Recombinant Gene Products Group, International Centre for Genetic Engineering and Biotechnology, New Delhi, India.

Dengue is a viral pandemic caused by four dengue virus serotypes (DENV-1, 2, 3, and 4) transmitted by Aedes mosquitoes. Reportedly, there has been a 2-fold increase in dengue cases every decade. An efficacious tetravalent vaccine, which can provide long-term immunity against all four serotypes in all target populations, is still unavailable. Despite the progress being made in the live virus-based dengue vaccines, the World Health Organization strongly recommends the development of alternative approaches for safe, affordable, and efficacious dengue vaccine candidates. We have explored virus-like particles (VLPs)-based nonreplicating subunit vaccine approach and have developed recombinant envelope ectodomains of DENV-1, 2, and 3 expressed in Pichia pastoris These self-assembled into VLPs without pre-membrane (prM) protein, which limits the generation of enhancing antibodies, and elicited type-specific neutralizing antibodies against the respective serotype. Encouraged by these results, we have extended this work further by developing P. pastoris-expressed DENV-4 ectodomain (DENV-4 E) in this study, which was found to be glycosylated and assembled into spherical VLPs without prM, and displayed critical neutralizing epitopes on its surface. These VLPs were found to be immunogenic in mice and elicited DENV-4-specific neutralizing antibodies, which were predominantly directed against envelope domain III, implicated in host-receptor recognition and virus entry. These observations underscore the potential of VLP-based nonreplicative vaccine approach as a means to develop a safe, efficacious, and tetravalent dengue subunit vaccine. This work paves the way for the evaluation of a DENV E-based tetravalent dengue vaccine candidate, as an alternative to live virus-based dengue vaccines.
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http://dx.doi.org/10.4269/ajtmh.16-0503DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5239678PMC
January 2017

Investigational drugs in early development for treating dengue infection.

Expert Opin Investig Drugs 2016 Sep 24;25(9):1059-69. Epub 2016 Jun 24.

a Department of Biological Sciences , Birla Institute of Technology and Science Pilani , Hyderabad , India.

Introduction: Dengue has emerged as the most significant arboviral disease of the current century. A drug for dengue is an urgent unmet need. As conventional drug discovery efforts have not produced any promising clinical candidates, there is a shift toward re-positioning pre-existing drugs for dengue to fast-track dengue drug development.

Areas Covered: This article provides an update on the current status of recently completed and ongoing dengue drug trials. All dengue drug trials described in this article were identified from a list of >230 trials that were returned upon searching the World Health Organization's International Clinical Trials Registry Platform web portal using the search term 'dengue' on December 31(st), 2015.

Expert Opinion: None of the handful of drugs tested so far has yielded encouraging results. Early trial experience has served to emphasize the challenge of drug testing in the short therapeutic time window available, the need for tools to predict 'high-risk' patients early on and the limitations of the existing pre-clinical model systems. Significant investment of efforts and resources is a must before the availability of a safe, effective and inexpensive dengue drug becomes a reality. Currently, supportive fluid therapy remains the only option available for dengue treatment.
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http://dx.doi.org/10.1080/13543784.2016.1201063DOI Listing
September 2016

Virus-like particles derived from Pichia pastoris-expressed dengue virus type 1 glycoprotein elicit homotypic virus-neutralizing envelope domain III-directed antibodies.

BMC Biotechnol 2016 06 14;16(1):50. Epub 2016 Jun 14.

Recombinant Gene Products Group, International Centre for Genetic Engineering & Biotechnology, Aruna Asaf Ali Marg, New Delhi, 110067, India.

Background: Four antigenically distinct serotypes (1-4) of dengue viruses (DENVs) cause dengue disease. Antibodies to any one DENV serotype have the potential to predispose an individual to more severe disease upon infection with a different DENV serotype. A dengue vaccine must elicit homotypic neutralizing antibodies to all four DENV serotypes to avoid the risk of such antibody-dependent enhancement in the vaccine recipient. This is a formidable challenge as evident from the lack of protective efficacy against DENV-2 by a tetravalent live attenuated dengue vaccine that has completed phase III trials recently. These trial data underscore the need to explore non-replicating subunit vaccine alternatives. Recently, using the methylotrophic yeast Pichia pastoris, we showed that DENV-2 and DENV-3 envelope (E) glycoproteins, expressed in absence of prM, implicated in causing severe dengue disease, self-assemble into virus-like particles (VLPs), which elicit predominantly virus-neutralizing antibodies and confer significant protection against lethal DENV challenge in an animal model. The current study extends this work to a third DENV serotype.

Results: We cloned and expressed DENV-1 E antigen in P. pastoris, and purified it to near homogeneity. Recombinant DENV-1 E underwent post-translational processing, namely, signal peptide cleavage and glycosylation. Purified DENV-1 E self-assembled into stable VLPs, based on electron microscopy and dynamic light scattering analysis. Epitope mapping with monoclonal antibodies revealed that the VLPs retained the overall antigenic integrity of the virion particles despite the absence of prM. Subtle changes accompanied the efficient display of E domain III (EDIII), which contains type-specific neutralizing epitopes. These VLPs were immunogenic, eliciting predominantly homotypic EDIII-directed DENV-1-specific neutralizing antibodies.

Conclusions: This work demonstrates the inherent potential of P. pastoris-expressed DENV-1 E glycoprotein to self-assemble into VLPs eliciting predominantly homotypic neutralizing antibodies. This work justifies an investigation of the last remaining serotype, namely, DENV-4, to assess if it also shares the desirable vaccine potential manifested by the remaining three DENV serotypes. Such efforts could make it possible to envisage the development of a tetravalent dengue vaccine based on VLPs of P. pastoris-expressed E glycoproteins of the four DENV serotypes.
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http://dx.doi.org/10.1186/s12896-016-0280-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4908714PMC
June 2016

Inhibition of preS1-hepatocyte interaction by an array of recombinant human antibodies from naturally recovered individuals.

Sci Rep 2016 Feb 18;6:21240. Epub 2016 Feb 18.

Department of Biochemistry, All India Institute of Medical Sciences, New Delhi, India.

Neutralizing monoclonal antibodies are being found to be increasingly useful in viral infections. In hepatitis B infection, antibodies are proven to be useful for passive prophylaxis. The preS1 region (21-47a.a.) of HBV contains the viral hepatocyte-binding domain crucial for its attachment and infection of hepatocytes. Antibodies against this region are neutralizing and are best suited for immune-based neutralization of HBV, especially in view of their not recognizing decoy particles. Anti-preS1 (21-47a.a.) antibodies are present in serum of spontaneously recovered individuals. We generated a phage-displayed scFv library using circulating lymphocytes from these individuals and selected four preS1-peptide specific scFvs with markedly distinct sequences from this library. All the antibodies recognized the blood-derived and recombinant preS1 containing antigens. Each scFv showed a discrete binding signature, interacting with different amino acids within the preS1-peptide region. Ability to prevent binding of the preS1 protein (N-terminus 60a.a.) to HepG2 cells stably expressing hNTCP (HepG2-hNTCP-C4 cells), the HBV receptor on human hepatocytes was taken as a surrogate marker for neutralizing capacity. These antibodies inhibited preS1-hepatocyte interaction individually and even better in combination. Such a combination of potentially neutralizing recombinant antibodies with defined specificities could be used for preventing/managing HBV infections, including those by possible escape mutants.
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http://dx.doi.org/10.1038/srep21240DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4758072PMC
February 2016

Casamino acids facilitate the secretion of recombinant dengue virus serotype-3 envelope domain III in Pichia pastoris.

BMC Biotechnol 2016 Feb 4;16:12. Epub 2016 Feb 4.

Centre for Biodesign and Diagnostics, Translational Health Science and Technology Institute, NCR Biotech Science Cluster, Faridabad, Haryana, India.

Background: Dengue is a viral disease spread to humans by mosquitoes. Notably, there are four serotypes of dengue viruses (DENV) that places ~40 % of the global population at risk of infection. However, lack of a suitable drug or a preventive vaccine exacerbates the matter further. Envelope domain-III (EDIII) antigen of dengue virus (DENV) has garnered much attention as a promising vaccine candidate for dengue, in addition to its use as a diagnostic intermediate. Hence developing a method for efficient production of high quality recombinant EDIII is important for research and industrial purpose.

Results: In this work, a Pichia pastoris system was optimized for the secretory over-expression of DENV serotype-3 EDIII under the control of methanol inducible AOX1 promoter. Temperature alone had a significant impact upon the amount of secretory EDIII, with 2.5-fold increase upon reducing the induction temperature from 30 to 20 °C. However surprisingly, supplementation of culture media with Casamino acids (CA), further augmented secretory EDIII titer, with a concomitant drop of intracellular EDIII levels at both temperatures. Though, reduction in intracellular retention of EDIII was more prominent at 20 °C than 30 °C. This suggests that CA supplementation facilitates overexpressing P. pastoris cells to secrete more EDIII by reducing the proportion retained intracellularly. Moreover, a bell-shaped correlation was observed between CA concentration and secretory EDIII titer. The maximum EDIII expression level of 187 mg/L was achieved under shake flask conditions with induction at 20 °C in the presence of 1 % CA. The overall increase in EDIII titer was ~9-fold compared to un-optimized conditions. Notably, mouse immune-sera, generated using this purified EDIII antigen, efficiently neutralized the DENV.

Conclusions: The strategy described herein could enable fulfilling the mounting demand for recombinant EDIII as well as lay direction to future studies on secretory expression of recombinant proteins in P. pastoris with CA as a media supplement.
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http://dx.doi.org/10.1186/s12896-016-0243-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4743106PMC
February 2016

Europium nanoparticle-based simple to perform dry-reagent immunoassay for the detection of hepatitis B surface antigen.

J Virol Methods 2016 Mar 4;229:66-9. Epub 2016 Jan 4.

Department of Biotechnology, University of Turku, Turku, Finland.

Hepatitis B infection, caused by hepatitis B virus (HBV), presents a huge global health burden. Serological diagnosis of HBV mainly relies on the detection of hepatitis B surface antigen (HBsAg). Although there are high sensitivity commercial HBsAg enzyme immunoassays (EIAs) available, many low-resource laboratories lacking trained technicians continue to use rapid point-of-care assays with low sensitivities for HBsAg detection, due to their simplicity to operate. We developed a time-resolved fluorometric dry-reagent HBsAg immunoassay which meets the detection limit of high sensitivity EIAs but is simple to operate. To develop the assay, anti-HBsAg monoclonal antibody coated on europium nanoparticles was dried atop of biotinylated anti-HBsAg polyclonal antibody immobilized on streptavidin-coated microtiter wells. To test a sample in dry-reagent assay, serum sample and assay buffer were added to the wells, incubated, washed and europium signals were measured. The assay showed a detection limit of 0.25 ng/ml using HBsAg spiked in serum sample. When evaluated with 24 HBV positive and 37 negative serum samples, assay showed 100% sensitivity and specificity. Assay wells are stable for at least 26 weeks when stored at 4°C, and can tolerate elevated temperatures of up to 35°C for two weeks. The developed assay has high potential to be used in low-resource laboratories.
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http://dx.doi.org/10.1016/j.jviromet.2016.01.001DOI Listing
March 2016

Array-in-well platform-based multiplex assay for the simultaneous detection of anti-HIV- and treponemal-antibodies, and Hepatitis B surface antigen.

J Immunol Methods 2016 Feb 19;429:21-7. Epub 2015 Dec 19.

Department of Biotechnology, University of Turku, Turku, Finland.

Multiplex assays detecting sets of related clinical analytes simultaneously can save considerable amount of time and resources. Array-in-well (AIW) is a powerful platform for the multiplex detection of different analytes where microarrays can be printed at the bottom of microtiter wells, thus combining the potential of microarrays with the ease of handling microtiter wells. We have developed a single-step AIW assay for the simultaneous screening of HIV, Treponema pallidum subspecies pallidum (causing syphilis) and Hepatitis B virus infections targeting the specific detection of anti-HIV- and treponemal-antibodies and Hepatitis B surface antigen (HBsAg), respectively, using two different fluorescent label technologies i.e. DyLight 633 and europium nanoparticle. Double-antigen assay formats were used for anti-HIV- and treponemal-antibody detection that can simultaneously detect both IgG and IgM, and thus reduce the window period of detection. AIW assay was evaluated with well characterized serum/plasma samples (n=111), and the qualitative results were in near complete agreement with those of the reference assays. The AIW assay exhibited 100% sensitivities for all three analytes, and 100% specificities for anti-HIV antibodies and HBsAg, and 98.6% specificity for treponemal antibodies. The limit of detection of HBsAg in AIW assay was 0.18 ng/ml. This high performing AIW assay has the potential to be used as a multiplex screening test for these three infections.
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http://dx.doi.org/10.1016/j.jim.2015.12.007DOI Listing
February 2016

Cissampelos pareira Linn: Natural Source of Potent Antiviral Activity against All Four Dengue Virus Serotypes.

PLoS Negl Trop Dis 2015 Dec 28;9(12):e0004255. Epub 2015 Dec 28.

Recombinant Gene Products Group, International Centre for Genetic Engineering and Biotechnology, New Delhi, India.

Background: Dengue, a mosquito-borne viral disease, poses a significant global public health risk. In tropical countries such as India where periodic dengue outbreaks can be correlated to the high prevalence of the mosquito vector, circulation of all four dengue viruses (DENVs) and the high population density, a drug for dengue is being increasingly recognized as an unmet public health need.

Methodology/principal Findings: Using the knowledge of traditional Indian medicine, Ayurveda, we developed a systematic bioassay-guided screening approach to explore the indigenous herbal bio-resource to identify plants with pan-DENV inhibitory activity. Our results show that the alcoholic extract of Cissampelos pariera Linn (Cipa extract) was a potent inhibitor of all four DENVs in cell-based assays, assessed in terms of viral NS1 antigen secretion using ELISA, as well as viral replication, based on plaque assays. Virus yield reduction assays showed that Cipa extract could decrease viral titers by an order of magnitude. The extract conferred statistically significant protection against DENV infection using the AG129 mouse model. A preliminary evaluation of the clinical relevance of Cipa extract showed that it had no adverse effects on platelet counts and RBC viability. In addition to inherent antipyretic activity in Wistar rats, it possessed the ability to down-regulate the production of TNF-α, a cytokine implicated in severe dengue disease. Importantly, it showed no evidence of toxicity in Wistar rats, when administered at doses as high as 2g/Kg body weight for up to 1 week.

Conclusions/significance: Our findings above, taken in the context of the human safety of Cipa, based on its use in Indian traditional medicine, warrant further work to explore Cipa as a source for the development of an inexpensive herbal formulation for dengue therapy. This may be of practical relevance to a dengue-endemic resource-poor country such as India.
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http://dx.doi.org/10.1371/journal.pntd.0004255DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4692392PMC
December 2015

Anti-HCV immunoassays based on a multiepitope antigen and fluorescent lanthanide chelate reporters.

J Virol Methods 2016 Feb 23;228:67-73. Epub 2015 Nov 23.

Department of Biotechnology, University of Turku, Turku, Finland.

There is a need for simple to produce immunoassays for hepatitis C virus (HCV) antibody capable of detecting all genotypes worldwide. Current commonly used third generation immunoassays use three to six separate recombinant proteins or synthetic peptides. We have developed and expressed in Escherichia coli a single recombinant antigen incorporating epitopes from different HCV proteins. This multiepitope protein (MEP) was used to develop two types of HCV antibody immunoassays: a traditional antibody immunoassay using a labeled secondary antibody (indirect assay) and a double-antigen assay with the same MEP used as capture binder and labeled binder. The secondary antibody assay was evaluated with 171 serum/plasma samples and double-antigen assay with 148 samples. These samples included an in-house patient sample panel, two panels of samples with different HCV genotypes and a seroconversion panel. The secondary antibody immunoassay showed 95.6% sensitivity and 100% specificity while the double-antigen assay showed 91.4% sensitivity and 100% specificity. Both assays detected samples from all six HCV genotypes. The results showed that combining a low-cost recombinant MEP binder antigen with a high sensitivity fluorescent lanthanide reporter can provide a sensitive and specific immunoassay for HCV serology. The results also showed that the sensitivity of HCV double-antigen assays may suffer from the low avidity immune response of acute infections.
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http://dx.doi.org/10.1016/j.jviromet.2015.11.015DOI Listing
February 2016

All-in-one dry-reagent time-resolved immunofluorometric assay for the rapid detection of HIV-1 and -2 infections.

J Virol Methods 2015 Dec 22;226:52-9. Epub 2015 Oct 22.

Department of Biotechnology, University of Turku, Turku, Finland.

An all-in-one (AIO) dry-reagent time-resolved fluorometric immunoassay that requires minimal liquid handling was developed for the detection of anti-HIV-1 and -2 antibodies. To prepare the AIO wells, in vivo biotinylated capture antigens (r-Bio-HIV-1env and r-Bio-HIV-2env) were immobilized on streptavidin-coated microtitration wells and Eu(III) chelate labelled non-biotinylated tracer antigens [r-HIV-1env-Eu(III) and r-HIV-2env-Eu(III)] were dried in stable form in the same wells. The HIV AIO assay was evaluated with serum/plasma samples (n=148) from in-house and commercial panels at two different incubation times of 15 min and 1h. The overall sensitivity of the AIO assay was 98.6% and specificity was 100% for both the incubation times. The AIO assay can accept whole blood matrix. This assay is envisioned to fill the gap between the rapid point-of-care assays and traditional enzyme immunoassays (EIA) in terms of complexity and turnaround time, without compromising the performance.
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http://dx.doi.org/10.1016/j.jviromet.2015.10.004DOI Listing
December 2015

Pichia pastoris-expressed dengue 3 envelope-based virus-like particles elicit predominantly domain III-focused high titer neutralizing antibodies.

Front Microbiol 2015 23;6:1005. Epub 2015 Sep 23.

Recombinant Gene Products Group, International Centre for Genetic Engineering and Biotechnology, New Delhi India ; Translational Health Science and Technology Institute, NCR Biotech Science Cluster, Faridabad India ; Department of Pediatrics, Emory University School of Medicine, Atlanta, GA USA.

Dengue poses a serious public health risk to nearly half the global population. It causes ~400 million infections annually and is considered to be one of the fastest spreading vector-borne diseases. Four distinct serotypes of dengue viruses (DENV-1, -2, -3, and -4) cause dengue disease, which may be either mild or extremely severe. Antibody-dependent enhancement (ADE), by pre-existing cross-reactive antibodies, is considered to be the major mechanism underlying severe disease. This mandates that a preventive vaccine must confer simultaneous and durable immunity to each of the four prevalent DENV serotypes. Recently, we used Pichia pastoris, to express recombinant DENV-2 E ectodomain, and found that it assembled into virus-like particles (VLPs), in the absence of prM, implicated in the elicitation of ADE-mediating antibodies. These VLPs elicited predominantly type-specific neutralizing antibodies that conferred significant protection against lethal DENV-2 challenge, in a mouse model. The current work is an extension of this approach to develop prM-lacking DENV-3 E VLPs. Our data reveal that P. pastoris-produced DENV-3 E VLPs not only preserve the antigenic integrity of the major neutralizing epitopes, but also elicit potent DENV-3 virus-neutralizing antibodies. Further, these neutralizing antibodies appear to be exclusively directed toward domain III of the DENV-3 E VLPs. Significantly, they also lack discernible ADE potential toward heterotypic DENVs. Taken together with the high productivity of the P. pastoris expression system, this approach could potentially pave the way toward developing a DENV E-based, inexpensive, safe, and efficacious tetravalent sub-unit vaccine, for use in resource-poor dengue endemic countries.
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http://dx.doi.org/10.3389/fmicb.2015.01005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4585145PMC
October 2015

A repertoire of high-affinity monoclonal antibodies specific to S. typhi: as potential candidate for improved typhoid diagnostic.

Immunol Res 2015 Jul;62(3):325-40

Centre for Bio-design and Diagnostics, Translational Health Science and Technology Institute, NCR Biotech Science Cluster, 3rd Milestone, Faridabad-Gurgaon Expressway, Faridabad, 121001, Haryana, India.

Typhoid fever is a significant global health problem with highest burden on the developing world. The severity of typhoid is often underestimated, and currently available serological diagnostic assays are inadequate due to lack in requisite sensitivity and specificity. This underlines an absolute need to develop a reliable and accurate diagnostics that would benefit long-term disease control and treatment and to understand the real disease burden. Here, we have utilized flagellin protein of S. typhi that is surface accessible, abundantly expressed, and highly immunogenic, for developing immunodiagnostic tests. Flagellin monomers are composed of conserved amino-terminal and carboxy-terminal, and serovar-specific middle region. We have generated a panel of murine monoclonal antibodies (mAbs) against the middle region of flagellin, purified from large culture of S. typhi to ensure its native conformation. These mAbs showed unique specificity and very high affinity toward S. typhi flagellin without showing any cross-reactivity with other serovars. Genetic analysis of mAbs also revealed high frequency of somatic mutation due to antigenic selection process across variable region to achieve high binding affinity. These antibodies also displayed stable binding in stringent reaction conditions for antigen-antibody interactions, like DMSO, urea, KSCN, guanidinium HCl, and extremes of pH. One of the mAbs potentially reversed the TLR5-mediated immune response, in vitro by inhibiting TLR5-flagellin interaction. In our study, binding of these mAbs to flagellin, with high affinity, present on bacterial surface, as well as in soluble form, validates their potential use in developing improved diagnostics with significantly higher sensitivity and specificity.
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http://dx.doi.org/10.1007/s12026-015-8663-zDOI Listing
July 2015

Drugs for dengue: a patent review (2010-2014).

Expert Opin Ther Pat 2014 Nov 6;24(11):1171-84. Epub 2014 Oct 6.

Birla Institute of Technology and Science Pilani, Department of Biological Sciences , Hyderabad Campus, Jawahar Nagar, Shameerpet Mandal, Ranga Reddy District, Hyderabad-500078 , India +91 40 66303631 ; +91 40 66303998 ; ,

Introduction: Almost half the global population is estimated to be at risk of contracting dengue infection. Of the 400 million infections estimated to occur annually, 4 million can be potentially life-threatening leading to vascular leakage and shock. The only treatment available to severe dengue patients is fluid replacement therapy and supportive care. A drug for treating dengue is an urgent need.

Areas Covered: This article endeavors to provide an overview of the experimental dengue drugs being developed around the world as reflected in the recent patent literature spanning the last few years (2010-2014).

Expert Opinion: Dengue drug development is essentially in its infancy and currently hobbled by multiple factors including a poor understanding of the molecular mechanism of severe disease and lack of reliable small animal model for preclinical drug evaluation. More intense R&D coupled to setting up product development partnerships to facilitate the efficient movement of a drug molecule from the laboratory to the clinic is needed to make antiviral therapy for dengue a reality in the coming future.
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http://dx.doi.org/10.1517/13543776.2014.967212DOI Listing
November 2014