Biomedical subjects

Navin Khanna

Publications and source records attributed to Navin Khanna.

14 recordsLinked to original sources

PubMed2006

Induction of neutralizing antibodies and T cell responses by dengue virus type 2 envelope domain III encoded by plasmid and adenoviral vectors.

Dengue is a re-emerging public health problem in many developing nations. There is neither a specific antiviral therapy to treat nor a licensed vaccine to prevent dengue infections. In recent years, the carboxy-terminal region of the major dengue virion envelope (E) protein, known as domain III, has emerged as a significant sub-unit vaccine candidate. In this study, we created a recombinant adenovirus capable of expressing the E domain III (EDIII) of dengue virus type 2 (DEN-2) and tested it in combination with a plasmid encoding the same domain to determine its potential as a possible dengue vaccine candidate. We examined the relative efficacies of plasmid prime/Ad boost (P/A) and Ad prime/plasmid boost (A/P) regimens in eliciting DEN-2 virus-specific immune responses. Both regimens resulted in the induction of antibodies that specifically bound to and neutralized the infectivity of DEN-2 virus. Splenocytes from immunized mice, stimulated in vitro, manifested a significant proliferative response accompanied by the production of high levels of interferon-gamma, but moderately elevated levels of interleukin-4, indicative of a predominantly Th1 type response. Based on a comparison of the parameters investigated, the immune response induced by the A/P regimen appeared to be relatively more potent. Our data suggest that EDIII may be valuable in the efforts to develop plasmid- and Ad-vectored dengue vaccines.

Adenoviridae↗

PubMed2006

A synthetic dengue virus antigen elicits enhanced antibody titers when linked to, but not mixed with, Mycobacterium tuberculosis HSP70 domain II.

Domain II of Mycobacterium tuberculosis (Mtb) heat shock protein 70 (HSP70), spanning amino acid residues 161-370, was covalently linked to a recently described synthetic dengue virus antigen to study the influence of the former on the immunogenicity of the latter. Using an enzyme-linked immunosorbent assay, dengue antigen-specific antibody titers elicited by the fusion protein in Balb/c mice were an order of magnitude higher than those elicited by either the synthetic dengue antigen alone or a physical mixture of the dengue antigen plus Mtb HSP70 domain II protein. Our data demonstrate that (i) Mtb HSP70 domain II is capable of potentiating B-cell response and (ii) it should be covalently linked to the target antigen to do so.

Animals↗

PubMed2006

Generation and characterization of a high-affinity chimaeric antibody against hepatitis B surface antigen.

Antibody against HBsAg (hepatitis B surface antigen) is advocated for the passive immunotherapy in certain cases of hepatitis B infections. A recombinant monoclonal antibody against HBsAg would offer several advantages over the currently used polyclonal human hepatitis B immunoglobulin. 5S is a mouse monoclonal antibody that binds to HBsAg with very high affinity. However, this mouse antibody cannot be used for therapeutic purposes, as it may elicit antimouse immune responses. Chimaerization, by replacing mouse constant domains with human counterparts, can reduce the immunogenicity of this molecule. We have cloned the V(H) (heavy-chain variable region) and V(L) (light-chain variable region) genes of this mouse antibody, and fused them with C(H)1 (heavy-chain constant domain 1) of human IgG1 and C(L) (light-chain constant domain) of human kappa chain respectively. These chimaeric genes were cloned into a mammalian expression vector (pFab-CMV), which has a modular cassette coding for part of the hinge, C(H)2 and C(H)3 of human IgG1. The recombinant construct was transfected in CHO (Chinese-hamster ovary) cells to generate a stable transfectoma. The resulting transfectoma was maintained in a serum-free medium and the full-length chimaeric anti-HBsAg antibody was purified from the culture supernatant. The yield of the purified chimaeric antibody was moderate ( approximately 5.5 mg/l). We further characterized the chimaeric antibody using several in vitro techniques. It was observed that the chimaeric molecule was glycosylated and expressed in the expected heterodimeric form. This chimaeric antibody has very high affinity and specificity, similar to that of the original mouse monoclonal antibody.

Animals↗

PubMed2006

Generation and characterization of a single-gene mouse-human chimeric antibody against hepatitis B surface antigen.

BACKGROUND: Antibody against hepatitis B surface antigen (HBsAg) is used for passive immunotherapy in certain cases of hepatitis B infection. The authors have earlier reported a high-affinity mouse monoclonal (5S) against HBsAg. However, this mouse antibody cannot be used for therapeutic purposes because it may elicit antimouse immune responses. Chimerization by replacing mouse constant domains with human ones can reduce the immunogenicity of this antibody. METHODS: A single-chain variable fragment (scFv), derived from the mouse monoclonal 5S, was fused with the fragment crystallisable (Fc) fragment of human IgG1. The scFv region is expected to bind to the antigen, whereas the Fc fragment can provide the effector functions required for virus neutralization. This chimeric molecule was expressed in Chinese hamster ovary (CHO) cells in serum-free medium. It was purified by affinity chromatography and characterized by in vitro binding studies. RESULTS: Purification and characterization indicated that this chimeric scFv-Fc fusion protein is secreted as a disulfide-linked, glycosylated, homodimeric molecule. The yield of the purified chimeric antibody was approximately 4.6 mg/L. In vitro analyses confirmed that this chimeric molecule retained the high affinity and specificity of the original mouse monoclonal. CONCLUSION: Because it is a single-gene product, this chimeric scFv-Fc has the advantage of stable expression. Being chimeric and bivalent, it is expected to be less immunogenic and therefore suitable for further in vivo studies on virus neutralization.

Animals↗

PubMed2006

Recombinant multiepitope protein for early detection of dengue infections.

Dengue fever is a mosquito-borne viral disease prevalent mainly in tropical countries. As the clinical manifestations of dengue are not very unique, laboratory diagnosis is crucial in identifying cases of dengue infection. Detection of dengue infection based on the identification of antidengue antibodies has emerged as a practical and reliable means of diagnosing dengue fever. We recently developed a customized recombinant dengue multiepitope protein (r-DME-G) that can specifically detect the immunoglobulin G (IgG) class of antidengue antibodies in patient sera. Using this strategy, we have now created another dengue multiepitope protein, r-DME-M, with specificity for the IgM class of antidengue antibodies. A synthetic gene encoding the r-DME-M protein was expressed as a maltose-binding protein fusion in Escherichia coli. The recombinant protein was purified in a single affinity chromatographic step to obtain yields of approximately 15 mg purified protein/liter of culture. The purified protein was used to develop an in-house IgM enzyme-linked immunosorbent assay (ELISA) and tested using a panel of 172 patient sera characterized using the commercially available Dengue Duo rapid strip test from PanBio, Australia. The IgM ELISA results showed that the r-DME-M protein not only recognized all IgM(+) samples identified by the PanBio test but also identified samples missed by the latter test. We also successfully adapted the r-DME-M protein to a rapid strip test format. This approach of creating customized antigens coupled to overexpression in E. coli and simple purification offers a promising alternative option to dengue diagnosis with the potential to circumvent the drawbacks of the whole virus antigen-based commercial kits.

Amino Acid Sequence↗

PubMed2006

Induction of neutralizing antibodies specific to dengue virus serotypes 2 and 4 by a bivalent antigen composed of linked envelope domains III of these two serotypes.

There is no vaccine to prevent dengue fever, a mosquito-borne viral disease, caused by four serotypes of dengue viruses. In this study, which has been prompted by the emergence of dengue virus envelope domain III as a promising sub-unit vaccine candidate, we have examined the possibility of developing a chimeric bivalent antigen with the potential to elicit neutralizing antibodies against two serotypes simultaneously. We created a chimeric dengue antigen by splicing envelope domain IIIs of serotypes 2 and 4. It was expressed in Escherichia coli and purified to near homogeneity. This protein retains the antigenic identities of both its precursors. It elicited antibodies that could efficiently block host cell binding of both serotypes 2 and 4 of dengue virus and neutralize their infectivity (neutralizing antibody titers approximately 1:40 and ~1:80 for dengue virus serotypes 2 and 4, respectively). This work could be a forerunner to the development of a single envelope domain III-based tetravalent antigen.

Animals↗

PubMed2005

High affinity mouse-human chimeric Fab against hepatitis B surface antigen.

AIM: Passive immunotherapy using antibody against hepatitis B surface antigen (HBsAg) has been advocated in certain cases of Hepatitis B infection. We had earlier reported on the cloning and expression of a high affinity scFv derived from a mouse monoclonal (5S) against HBsAg. However this mouse antibody cannot be used for therapeutic purposes as it may elicit anti-mouse immune responses. Chimerization by replacing mouse constant domains with human ones can reduce the immunogenicity of this antibody. METHODS: We cloned the V(H) and V(L) genes of this mouse antibody, and fused them with CH1 domain of human IgG1 and C(L) domain of human kappa chain respectively. These chimeric genes were cloned into a phagemid vector. After initial screening using the phage display system, the chimeric Fab was expressed in soluble form in E. coli. RESULTS: The chimeric Fab was purified from the bacterial periplasmic extract. We characterized the chimeric Fab using several in vitro techniques and it was observed that the chimeric molecule retained the high affinity and specificity of the original mouse monoclonal. This chimeric antibody fragment was further expressed in different strains of E. coli to increase the yield. CONCLUSION: We have generated a mouse-human chimeric Fab against HBsAg without any significant loss in binding and epitope specificity. This chimeric Fab fragment can be further modified to generate a full-length chimeric antibody for therapeutic uses.

Animals↗

PubMed2005

The identification of immunodominant linear epitopes of dengue type 2 virus capsid and NS4a proteins using pin-bound peptides.

We have used multi-pin peptide synthesis strategy to identify B-cell epitopes on two small dengue virus proteins, capsid and NS4a. We have identified several linear, immunodominant epitopes on both these proteins. Almost all these epitopes mapped to regions predicted to be hydrophilic based on Kyte and Doolittle profiles. Of the capsid epitopes identified in this study, the most immunogenic ones mapped to the C-terminal alpha4 helix, which lies on the solvent-exposed surface of the capsid dimer. The capsid epitopes were dengue-specific in that they could recognize antibodies in dengue virus-, but not yellow fever virus (YFV)- or Japanese encephalitis virus (JEV)-immune sera. This study has demonstrated the presence of anti-NS4a antibodies in dengue-patient sera definitively, for the first time, using authentic NS4a-derived pin-bound peptides as capture antigens. All the NS4a epitopes mapped to the amino-terminal third of the NS4a molecule. Our study suggests that the immunodominant epitopes of these two dengue proteins might have the potential to be used as a part of a recombinant multi-epitope protein containing carefully chosen E and NS1 epitopes for the detection of dengue infections with a high degree of sensitivity and specificity.

Amino Acid Sequence↗

PubMed2005

A custom-designed recombinant multiepitope protein as a dengue diagnostic reagent.

Currently, dengue fever is the most important re-emerging mosquito-borne viral disease, with the major proportion of the target population residing in the developing countries of the world. In endemic areas, potentially fatal secondary dengue infections, characterized by high anti-dengue IgG antibody titers, are most common. Most currently available commercial dengue diagnostic kits rely on the use of whole virus antigens and are consequently associated with false positives due to serologic cross-reactivity, high cost of antigen production, and biohazard risk. This has prompted the need to develop an alternate antigen to replace the whole virus antigen in diagnostic tests. We have designed and expressed a novel recombinant protein antigen by assembling key immunodominant linear IgG-specific dengue virus epitopes, chosen on the basis of pepscan analysis, phage display, and computer predictions. The recombinant dengue multiepitope protein was expressed to high levels in Escherichia coli, purified in a single step, yielding >25 mg pure protein per liter culture. We developed an in-house enzyme-linked immunosorbent assay (ELISA) to detect anti-dengue antibodies in a panel of 20 patient sera using the purified recombinant dengue multiepitope protein as the capture antigen. The ELISA results were in excellent agreement with those obtained using a commercially available diagnostic test, Dengue Duo rapid strip test from PanBio, Australia. The high epitope density, careful choice of epitopes, and the use of E. coli system for expression, coupled to simple purification, jointly have the potential to lead to the development of an inexpensive diagnostic test with a high degree of sensitivity and specificity.

Amino Acid Sequence↗

PubMed2004

High-level expression and one-step purification of recombinant dengue virus type 2 envelope domain III protein in Escherichia coli.

Dengue virus infection poses a serious global public health threat for which there is currently no therapy or a licensed vaccine. The domain III of the dengue virus encoded envelope protein, which carries multiple conformation-dependent neutralizing epitopes, is critical for virus infectivity. We have expressed and purified recombinant domain III of dengue virus type-2 envelope, without the aid of a carrier protein in Escherichia coli. A 6x His tag was inserted at the N terminus to facilitate its one-step purification. The protein was overexpressed in the form of insoluble inclusion bodies, which were solubilized under highly denaturing conditions and then subjected to a previously optimized arginine-mediated renaturation protocol. We purified recombinant domain III protein to near homogeneity by Ni-NTA affinity chromatography and obtained yields of approximately 30 mg/L. The purified protein was recognized in Western analyses by monoclonal antibodies specific for the 6x His tag as well as the 3H5 neutralizing epitope known to reside in domain III. The authenticity of the recombinant protein was also verified in a sandwich ELISA designed to specifically and simultaneously identify the 6x His tag and the 3H5 epitope. In addition, murine and human polyclonal sera also recognized the recombinant protein. The in vitro refolded recombinant protein preparation was biologically functional. It could effectively protect cells in culture against dengue virus type-2 infection, apparently by blocking the virus from binding to host cells. This expression/purification strategy has the potential for inexpensive scale-up and may prove to be useful for dengue diagnostics and vaccine development efforts.

Animals↗

PubMed2004

Engineering, cloning, and expression of genes encoding the multimeric luteinizing-hormone-releasing hormone linked to T cell determinants in Escherichia coli.

Two synthetic genes were designed and engineered to encode for multimeric luteinizing-hormone-releasing hormone (LHRH) peptides linked to T cell determinants. These genes were cloned into the prokaryotic expression vectors under control of strong inducible promoters, to overexpress the multimeric LHRH peptides as recombinant proteins. Multimeric LHRH-T cell peptides were expressed as insoluble inclusion bodies in Escherichia coli cultures. Cell extracts containing the recombinant proteins showed immunoreactivity on Western blots with monoclonal antibody recognizing the native hormonal peptide. These gene constructs have potential applications in therapy of sex-steroid-hormone-dependent cancers.

Amino Acid Sequence↗

PubMed2003

Characterization and molecular modeling of a highly stable anti-Hepatitis B surface antigen scFv.

We raised a mouse monoclonal antibody (5S) against the 'a' epitope of the Hepatitis B surface antigen (HBsAg) by selecting for binding of the hybridoma supernatant in conditions that usually destabilize protein-protein interactions. This antibody, which was protective in an in vitro assay, had a high affinity with a relative dissociation constant in the nanomolar range. It also displayed stable binding to antigen in conditions that usually destabilize antigen-antibody interactions, like 30% DMSO, 8 M urea, 4 M NaCl, 1 M guanidium HCl and extremes of pH. The variable regions of the antibody were cloned and expressed as an single chain variable fragment (scFv) (A5). A5 had a relative affinity comparable to the mouse monoclonal and showed antigen binding in presence of 20% DMSO, 8 M urea and 3 M NaCl. It bound the antigen in the pH range of 6-8, though its tolerance for guanidium HCl was reduced. Sequence analysis demonstrated a significant increase in the frequency of somatic replacement mutations in CDRs over framework regions in the light but not in the heavy chain. A comparison of the molecular models of the variable regions of the 5S antibody and its germ-line precursor revealed that critical mutations in the heavy and light chains interface resulted in better inter-chain packing and in the movement of CDR H3 and CDR L1 from their germline positions, which may be important for better antigen binding. In addition to providing a reagent for neutralizing for the virus, such an antibody provides a model for the evolution of stable high affinity interaction during antibody maturation.

Amino Acid Sequence↗

PubMed2003

Replication-defective adenoviral vaccine vector for the induction of immune responses to dengue virus type 2.

A recombinant replication-defective adenovirus vector that can overexpress the ectodomain of the envelope protein of dengue virus type 2 (NGC strain) has been constructed. This virus was immunogenic in mice and elicited dengue virus type 2 specific B- and T-cell responses. Sera from immunized mice contained neutralizing antibodies that could specifically recognize dengue virus type 2 and neutralize its infectivity in vitro, indicating that this approach has the potential to confer protective immunity. In vitro stimulation of splenocytes (from immunized mice) with dengue virus type 2 resulted in a significant proliferative response accompanied by the production of high levels of gamma interferon but did not show significant changes in interleukin-4 levels. This is suggestive of a Th1-like response (considered to be important in the maturation of cytotoxic T lymphocytes that are essential for the elimination of virus-infected cells). The data show that adenovirus vectors offer a promising alternative strategy for the development of dengue virus vaccines.

Adenoviridae↗

PubMed2002

Recombinant dengue virus type 2 envelope/hepatitis B surface antigen hybrid protein expressed in Pichia pastoris can function as a bivalent immunogen.

A truncated version of the dengue virus type 2 envelope protein (Den2E) encoding the first 395 amino acid (aa) residues, and Den2E fused in-frame with the full-length 226-aa hepatitis B surface antigen (Den2E-HBsAg) protein were expressed in the methylotrophic yeast, Pichia pastoris. Both the recombinant proteins showed evidence of the capacity to form high molecular weight aggregates. Electron microscopic analysis of the purified proteins showed that while Den2E displayed an amorphous morphology, Den2E-HBsAg existed as well-structured virus-like particles (VLPs). Using immuno-gold electron microscopy, these VLPs were demonstrated to contain both components of the Den2E-HBsAg hybrid protein. Seroanalysis showed that the hybrid VLPs could function in vivo as bivalent immunogens, which could elicit immune responses directed against both components of the hybrid protein, as evidenced by ELISA, immunoprecipitation and immunofluorescence data.

Animals↗