Lubina Khan
All India Institute of Medical Sciences
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BMC Biotechnology | 2012
Rajesh Kumar; Raiees Andrabi; Ashutosh Tiwari; Somi Sankaran Prakash; Naveet Wig; Durgashree Dutta; Anurag Sankhyan; Lubina Khan; Subrata Sinha; Kalpana Luthra
BackgroundProduction of human monoclonal antibodies that exhibit broadly neutralizing activity is needed for preventing HIV-1 infection, however only a few such antibodies have been generated till date. Isolation of antibodies by the hybridoma technology is a cumbersome process with fewer yields. Further, the loss of unstable or slowly growing clones which may have unique binding specificities often occurs during cloning and propagation and the strongly positive clones are often lost. This has been avoided by the process described in this paper, wherein, by combining the strategy of EBV transformation and recombinant DNA technology, we constructed human single chain variable fragments (scFvs) against the third variable region (V3) of the clade C HIV-1 envelope.ResultsAn antigen specific phage library of 7000 clones was constructed from the enriched V3- positive antibody secreting EBV transformed cells. By ligation of the digested scFv DNA into phagemid vector and bio panning against the HIV-1 consensus C and B V3 peptides followed by random selection of 40 clones, we identified 15 clones that showed V3 reactivity in phage ELISA. DNA fingerprinting analysis and sequencing showed that 13 out of the 15 clones were distinct. Expression of the positive clones was tested by SDS-PAGE and Western blot. All the 13 anti-V3 scFvs showed cross-reactivity against both the clade C and B V3 peptides and did not show any reactivity against other unrelated peptides in ELISA. Preliminary neutralization assays indicated varying degrees of neutralization of clade C and B viruses. EBV transformation, followed by antigen selection of lines to identify specific binders, enabled the selection of phage from un-cloned lines for scFv generation, thus avoiding the problems of hybridoma technology. Moreover, as the clones were pretested for antigen binding, a comparatively small library sufficed for the selection of a considerable number of unique antigen binding phage. After selection, the phage clones were propagated in a clonal manner.ConclusionsThis strategy can be efficiently used and is cost effective for the generation of diverse recombinant antibodies. This is the first study to generate anti-V3 scFvs against HIV-1 Clade C.
PLOS ONE | 2015
Lubina Khan; Muzamil Ashraf Makhdoomi; Sanjeev Kumar; Ambili Nair; Raiees Andrabi; Brenda E. Clark; Kate Auyeung; Jayanta Bhattacharya; Madhu Vajpayee; Naveet Wig; Ralph Pantophlet; Kalpana Luthra
Dissecting antibody specificities in the plasma of HIV-1 infected individuals that develop broadly neutralizing antibodies (bNAbs) is likely to provide useful information for refining target epitopes for vaccine design. Several studies have reported CD4-binding site (CD4bs) antibodies as neutralization determinants in the plasma of subtype B-infected individuals; however there is little information on the prevalence of CD4bs specificities in HIV-infected individuals in India. Here, we report on the presence of CD4bs antibodies and their contribution to virus neutralization in the plasma from a cohort of HIV-1 infected Indian individuals. Plasma from 11 of the 140 HIV-1 infected individuals (7.9%) studied here exhibited cross-neutralization activity against a panel of subtype B and C viruses. Analyses of these 11 plasma samples for the presence of CD4bs antibodies using two CD4bs-selective probes (antigenically resurfaced HXB2gp120 core protein RSC3 and hyperglycosylated JRFLgp120 mutant ΔN2mCHO) revealed that five (AIIMS 617, 619, 627, 642, 660) contained RSC3-reactive plasma antibodies and only one (AIIMS 660) contained ΔN2mCHO-reactive antibodies. Plasma antibody depletion and competition experiments confirmed that the neutralizing activity in the AIIMS 660 plasma was dependent on CD4bs antibodies. To the best of our knowledge, this is the first study to report specifically on the presence of CD4bs antibodies in the plasma of a cohort of HIV-1 infected Indian donors. The identification of CD4bs dependent neutralizing antibodies in an HIV-1 infected Indian donor is a salient finding of this study and is supportive of ongoing efforts to induce similar antibodies by immunization.
Scientific Reports | 2017
Lubina Khan; Rajesh Kumar; Hilal Ahmad Parray; Muzamil Ashraf Makhdoomi; Sanjeev Kumar; Heena Aggarwal; Madhav Mohata; Abdul Wahid Hussain; Raksha Das; Raghavan Varadarajan; Jayanta Bhattacharya; Madhu Vajpayee; Murugavel Kg; Suniti Solomon; Subrata Sinha; Kalpana Luthra
More than 50% of HIV-1 infection globally is caused by subtype_C viruses. Majority of the broadly neutralizing antibodies (bnAbs) targeting HIV-1 have been isolated from non-subtype_C infected donors. Mapping the epitope specificities of bnAbs provides useful information for vaccine design. Recombinant antibody technology enables generation of a large repertoire of monoclonals with diverse specificities. We constructed a phage recombinant single chain variable fragment (scFv) library with a diversity of 7.8 × 108 clones, using a novel strategy of pooling peripheral blood mononuclear cells (PBMCs) of six select HIV-1 chronically infected Indian donors whose plasma antibodies exhibited potent cross neutralization efficiency. The library was panned and screened by phage ELISA using trimeric recombinant proteins to identify viral envelope specific clones. Three scFv monoclonals D11, C11 and 1F6 selected from the library cross neutralized subtypes A, B and C viruses at concentrations ranging from 0.09 μg/mL to 100 μg/mL. The D11 and 1F6 scFvs competed with mAbs b12 and VRC01 demonstrating CD4bs specificity, while C11 demonstrated N332 specificity. This is the first study to identify cross neutralizing scFv monoclonals with CD4bs and N332 glycan specificities from India. Cross neutralizing anti-HIV-1 human scFv monoclonals can be potential candidates for passive immunotherapy and for guiding immunogen design.
Journal of General Virology | 2017
Muzamil Ashraf Makhdoomi; Lubina Khan; Sanjeev Kumar; Heena Aggarwal; Ravinder Singh; Rakesh Lodha; Mohit Singla; Bimal K. Das; Sushil K. Kabra; Kalpana Luthra
Delineating the factors leading to the development of broadly neutralizing antibodies (bnAbs) during natural HIV-1 infection and dissecting their epitope specificities generates useful information for vaccine design. This is the first longitudinal study to assess the plasma-neutralizing antibody response and neutralizing determinants in HIV-1-infected children from India. We enrolled 26 and followed up 20 antiretroviral therapy (ART)-naïve, asymptomatic, chronic HIV-1-infected children. Five (19.2 %) baseline and 10 (50 %) follow-up plasma samples neutralized ≥50 % of subtypes A, B and C tier 2 viruses at an ID50 titre ≥150. A modest improvement in neutralization breadth and potency was observed with time. At baseline, subtype C-specific neutralization predominated (P=0.026); interestingly, follow-up samples exhibited cross-neutralizing activity. Epitope mapping revealed V3C reactive antibodies with significantly increased Max50 binding titres in follow-up samples from five infected children; patient #4s plasma antibodies exhibited V3-directed neutralization. A salient observation was the presence of CD4 binding site (CD4bs)-specific NAbs in patient #18 that improved with time (1.76-fold). The RSC3 wild-type (RSC3WT) protein-depleted plasma eluate of patient #18 demonstrated a more than 50% ID50 decrease in neutralization capacity against five HIV-1 pseudoviruses. Further, the presence of CD4bs-neutralizing determinants in patient #18s plasma was confirmed by the neutralizing activity demonstrated by the CD4bs-directed IgG fraction purified from this plasma, and competition with sCD4 against JRFLgp120, identifying this paediatric donor as a potential candidate for the isolation of CD4bs-directed bnAbs. Overall, we observed a relative increase in plasma-neutralizing activity with time in HIV-1-infected children, which suggests that the bnAbs evolve.
BMC Infectious Diseases | 2014
Sanjeev Kumar; Rajesh Kumar; Muzamil Ashraf Makhdoomi; Lubina Khan; Somi Sankaran Prakash; Mohit Singla; Rakesh Lodha; Sushil K. Kabra; Subrata Sinha; Kalpana Luthra
Methods Nine ART drug naive HIV-1 subtype c infected children were recruited. PBMCs were isolated from all the subjects and pooled. RNA was isolated and cDNA was synthesized followed by amplification of VH and VL chain genes and scFv construction. A human recombinant scFv phage display library of 108 clones was constructed. Diversity of the phage library was checked by DNA sequencing and biopanned with RSC3 core antigen. 60 random clones were screened by phage ELISA. Expression of the scFvs was assessed by SDS-PAGE and Western blotting.
Frontiers in Immunology | 2017
Heena Aggarwal; Lubina Khan; Omkar Chaudhary; Sanjeev Kumar; Muzamil Ashraf Makhdoomi; Ravinder Singh; Kanika Sharma; Nitesh Mishra; Rakesh Lodha; M. Srinivas; Bimal K. Das; Sushil K. Kabra; Kalpana Luthra
Several B cell defects are reported in HIV-1 infected individuals including variation in B cell subsets, polyclonal B cell activation and exhaustion, with broadly neutralizing antibodies elicited in less than 10–20% of the infected population. HIV-1 disease progression is faster in children than adults. B Lymphocyte Stimulator (BLyS), expressed on dendritic cells (DCs), is a key regulator of B cell homeostasis. Understanding how DCs influence B cell phenotype and functionality (viral neutralization), thereby HIV-1 disease outcome in infected children, is important to develop interventional strategies for restoration of B cell function. In this study, a total of 38 vertically transmitted HIV-1 infected antiretroviral therapy (ART) naïve children and 25 seronegative controls were recruited. Based on the CD4 counts and years post-infection, infected children were categorized as long-term non-progressors (LTNPs) (n = 20) and progressors (n = 18). Eight of these progressors were followed up at 6–12 months post-ART. Percentages (%) of DCs, B cell subsets, and expression of BLyS on DCs were analyzed by flow-cytometry. Plasma levels of B cell growth factors were measured by ELISA and viral neutralization activity was determined using TZM-bl assay. Lower (%) of myeloid DCs (mDCs), plasmacytoid DCs, and high expression of BLyS on mDCs were observed in HIV-1 infected progressors than seronegative controls. Progressors showed lower % of naive B cells, resting memory B cells and higher % of mature activated, tissue-like memory B cells as compared to seronegative controls. Higher plasma levels of IL-4, IL-6, IL-10, and IgA were observed in progressors vs. seronegative controls. Plasma levels of IgG were high in progressors and in LTNPs than seronegative controls, suggesting persistence of hypergammaglobulinemia at all stages of disease. High plasma levels of BLyS in progressors positively correlated with poor viral neutralizing activity. Interestingly on follow up, treatment naïve progressors, post-ART showed increase in resting memory B cells along with reduction in plasma BLyS levels that correlated with improvement in viral neutralization. This is the first study to demonstrate that reduction in plasma BLyS levels correlates with restoration of B cell function, in terms of viral neutralization in HIV-1-infected children.
Frontiers in Immunology | 2017
Sanjeev Kumar; Rajesh Kumar; Lubina Khan; Muzamil Ashraf Makhdoomi; Madhav Mohata; Mudit Agarwal; Rakesh Lodha; Sushil K. Kabra; Subrata Sinha; Kalpana Luthra
Progression of human immunodeficiency virus type-1 (HIV-1) infection in children is faster than adults. HIV-1 subtype C is responsible for more than 50% of the infections globally and more than 90% infections in India. To date, there is no effective vaccine against HIV-1. Recent animal studies and human Phase I trials showed promising results of the protective effect of anti-HIV-1 broadly neutralizing antibodies (bnAbs). Interaction between CD4 binding site (CD4bs) on the HIV-1 envelope glycoprotein and CD4 receptor on the host immune cells is the primary event leading to HIV-1 infection. The CD4bs is a highly conserved region, comprised of a conformational epitope, and is a potential target of bnAbs such as VRC01 that is presently under human clinical trials. Recombinant scFvs can access masked epitopes due to their small size and have shown the potential to inhibit viral replication and neutralize a broad range of viruses. Pediatric viruses are resistant to many of the existing bnAbs isolated from adults. Therefore, in this study, pooled peripheral blood mononuclear cells from 9 chronically HIV-1 subtype C infected pediatric cross-neutralizers whose plasma antibodies exhibited potent and cross-neutralizing activity were used to construct a human anti-HIV-1 scFv phage library of 9 × 108 individual clones. Plasma mapping using CD4bs-specific probes identified the presence of CD4bs directed antibodies in 4 of these children. By extensive biopanning of the library with CD4bs-specific antigen RSC3 core protein, we identified two cross-neutralizing scFv monoclonals 2B10 and 2E4 demonstrating a neutralizing breadth and GMT of 77%, 17.9 µg/ml and 32%, 51.2 µg/ml, respectively, against a panel of 49 tier 1, 2 and 3 viruses. Both scFvs competed with anti-CD4bs bnAb VRC01 confirming their CD4bs epitope specificity. The 2B10 scFv was effective in neutralizing the 7 subtype C and subtype A pediatric viruses tested. Somatic hypermutations in the VH gene of scFvs (10.1–11.1%) is comparable with that of the adult antibodies. These cross-neutralizing CD4bs-directed scFvs can serve as potential reagents for passive immunotherapy. A combination of cross-neutralizing scFvs of diverse specificities with antiretroviral drugs may be effective in suppressing viremia at an early stage of HIV-1 infection and prevent disease progression.
Retrovirology | 2012
Rajesh Kumar; Raiees Andrabi; Ashutosh Tiwari; Somi Sankaran Prakash; Naveet Wig; Durgashree Dutta; Anurag Sankhyan; Lubina Khan; Subrata Sinha; Kalpana Luthra
Neutralizing antibodies are an important component of the humoral immune response directed against viral infections So far the few available anti HIV-1 broadly neutralizing antibodies have a limited breadth and potency against clade C viruses. More than 50% of the HIV-1 infections worldwide belong to clade C. Clade C is the most prevalent subtype in India.
BMC Infectious Diseases | 2012
Rajesh Kumar; Raiees Andrabi; Ashutosh Tiwari; Somi Sankaran Prakash; Naveet Wig; Durgashree Dutta; Anurag Sankhyan; Lubina Khan; Subrata Sinha; Kalpana Luthra
Methods A phage library of 7000 clones was constructed from a drug naive HIV-1 clade C infected Indian patient whose plasma exhibited high potential neutralizing potential against a panel of viruses and also displayed cross-reactive anti-V3 antibodies. PBMCs were isolated and EBV transformed. Cells (wells) producing anti-V3 antibodies were preselected with V3-CTB fusion protein and expanded. Total RNA was isolated and cDNA was constructed followed by VH and VL amplification. scFvs were constructed, cloned into phagemid vector and expressed in Escherichia coli. We assessed the expression of the scFvs by SDSPAGE and Western blotting. Specificity was examined by ELISA.
Applied Biochemistry and Biotechnology | 2018
Rajesh Kumar; Ruchi Kumari; Lubina Khan; Anurag Sankhyan; Hilal Ahmad Parray; Ashutosh Tiwari; Naveet Wig; Subrata Sinha; Kalpana Luthra
Recently conducted human phase- I trials showed protective effect of anti-HIV-1 broadly neutralizing antibodies (bnAbs). The V3 region of the HIV-1 envelope is highly conserved as it is the co-receptor binding site, and is highly immunogenic. Recombinant single-chain antibody fragments (scFvs) can serve as potential tools for construction of chimeric/bispecific antibodies that can target different epitopes on the HIV-1 envelope. Previously, we have constructed a V3 specific human scFv phage recombinant library by a combinational approach of Epstein–Barr virus (EBV) transformation and antigen (V3) preselection, using peripheral blood mononuclear cells (PBMCs), from a subtype C HIV-1 infected antiretroviral naive donor. In the present study, by biopanning this recombinant scFv phage library with V3B (subtype B) and V3C (subtype C) peptides, we identified unique cross reactive anti-V3 scFv monoclonals. These scFvs demonstrated cross-neutralizing activity when tested against subtype A, subtype B, and subtype C viruses. Further, molecular modeling of the anti-V3 scFvs with V3C and V3B peptides predicted their sites of interaction with the scFvs, providing insights for future immunogen design studies. A large collection of such monoclonal antibody fragments with diverse epitope specificities can be useful immunotherapeutic reagents along with antiretroviral drugs to prevent HIV-1 infection and disease progression.