Cross-neutralizing anti-HIV-1 human single chain variable fragments(scFvs) against CD4 binding site and N332 glycan identified from a recombinant phage library

Khan, Lubina; Kumar, Rajesh; Thiruvengadam, Ramachandran; Parray, Hilal Ahmad; Makhdoomi, Muzamil Ashraf; Kumar, Sanjeev; Aggarwal, Heena; Mohata, Madhav; Hussain, Abdul Wahid; Das, Raksha; Varadarajan, Raghavan; Bhattacharya, Jayanta; Vajpayee, Madhu; Murugavel, K; Solomon, Suniti; Sinha, Subrata; Luthra, Kalpana

doi:10.1038/srep45163

Cited by 19 publications

(22 citation statements)

References 67 publications

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“…Chronically HIV-1-infected children have been shown to have potent plasma bnAbs with diverse epitope specificities (5,6). In our pediatric cohort of antiretroviral-naive HIC-1 clade C (HIV-1C)-infected children previously characterized for plasma antibody responses (6,(13)(14)(15)(16)(17)(18), we identified a pair of chronically infected antiretroviral-naive HIV-1C-infected children, AIIMS_329 and AIIMS_330, defined herein as genetically identical twins by their identical HLA phenotypes, who had acquired the infection at birth by vertical transmission. Both twins AIIMS_329 and AIIMS_330 developed potent plasma neutralizing antibodies (nAbs), with the latter showing elite neutralizing activity since the first sampling.…”

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confidence: 99%

Viral Characteristics Associated with Maintenance of Elite Neutralizing Activity in Chronically HIV-1 Clade C-Infected Monozygotic Pediatric Twins

Mishra

Makhdoomi

Sharma

et al. 2019

J Virol

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Broad and potent neutralizing antibodies (bnAbs) with multiple epitope specificities evolve in HIV-1-infected children. Herein, we studied two antiretroviral-naive chronically HIV-1 clade C-infected monozygotic pediatric twins, AIIMS_329 and AIIMS_330, with potent plasma bnAbs. Elite plasma neutralizing activity was observed since the initial sampling at 78 months of age in AIIMS_330 and persisted throughout, while in AIIMS_329 it was seen at 90 months of age, after which the potency decreased over time. We evaluated potential viral characteristics associated with the varied immune profiles by generating single genome-amplified pseudoviruses. The AIIMS_329 viruses generated from the 90-month time point were neutralization sensitive to bnAbs and contemporaneous plasma antibodies, while viruses from the 112-month and 117-month time points were resistant to most bnAbs and contemporaneous plasma. AIIMS_329 viruses developed resistance to plasma neutralizing antibodies (nAbs) plausibly by N160 glycan loss and V1 and V4 loop lengthening. The viruses generated from AIIMS_330 (at 90 and 117 months) showed varied susceptibility to bnAbs and autologous contemporaneous plasma antibodies, while the viruses of the 112-month time point, at which the plasma nAb specificities mapped to the V2 glycan, V3 glycan, and CD4 binding site (CD4bs), were resistant to contemporaneous plasma antibodies as well as to most bnAbs. Chimeric viruses were constructed from 90-month-time-point PG9-sensitive AIIMS_329 and AIIMS_330 viruses with swapped V1V2 regions of their respective evolved viruses (at 112 and 117 months), which led to higher resistance to neutralization by PG9 and autologous plasma antibodies. We observed the evolution of a viral pool in the AIIMS_330 donor comprising plasma antibody neutralization-sensitive or -resistant diverse autologous viruses that may have contributed to the development and maintenance of elite neutralizing activity. IMPORTANCE Herein, we report the longitudinal development of bnAbs in a pair of chronically HIV-1 clade C-infected monozygotic pediatric twins, AIIMS_329 and AIIMS_330, who acquired the infection by vertical transmission. The plasma from both donors, sharing a similar genetic makeup and infecting virus, showed the evolvement of bnAbs targeting common epitopes in the V2 and V3 regions of the envelope, suggesting that bnAb development in these twins may perhaps be determined by specific sequences in the shared virus that can guide the development of immunogens aimed at eliciting V2 and V3 bNAbs. Characterization of the neutralization-sensitive and -resistant viruses coevolving with bNAbs in the contemporaneous AIIMS_330 plasma provides information toward understanding the viral alterations that may have contributed to the development of resistance to bnAbs. Further longitudinal studies in more monozygotic and dizygotic twin pairs will help in delineating the role of host and viral factors that may contribute to the development of bnAbs.

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confidence: 99%

Viral Characteristics Associated with Maintenance of Elite Neutralizing Activity in Chronically HIV-1 Clade C-Infected Monozygotic Pediatric Twins

Mishra

Makhdoomi

Sharma

et al. 2019

J Virol

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“…We did not determine the affinity of this interaction, but we infer from the phage ELISA and sequence analysis that these two antibodies were variable. Previous studies indicate that scFv antibodies are able to neutralize various viral infections, including influenza A virus (H5N1 subtype), human immunodeficiency virus 1, respiratory syncytial virus, rabies virus, and infectious bronchitis virus [43][44][45][46][47]. Notably, most scFv antibodies can target viral surface glycoproteins, which in turn play a role in blocking virus adherence to receptors of host cells.…”

Section: Discussionmentioning

confidence: 99%

Construction and characterization of porcine single-chain fragment variable antibodies that neutralize transmissible gastroenteritis virus in vitro

et al. 2019

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Transmissible gastroenteritis virus (TGEV) infection causes severe diarrhea in piglets and imposes a significant economic burden on pig farms. Single-chain fragment variable (scFv) antibodies effectively inhibit virus infection and could be a potential therapeutic reagent for preventing disease. In this study, a recombinant scFv antibody phage display library was constructed from peripheral blood lymphocytes of piglets infected with TGEV. The library was screened with four rounds of biopanning using purified TGEV antigen, and scFv antibodies that bound to TGEV were obtained. The scFv gene was subcloned into the pET-28a(+), and the constituted plasmid was introduced into Escherichia coli BL21 (DE3) for protein expression. All three scFv clones identified had neutralizing activity against TGEV. An immunofluorescence assay and western blot analysis demonstrated that two scFv antibodies reacted with the spike protein of TGEV. These results indicate that scFv antibodies provide protection against viral infection in vitro and may be a therapeutic candidate for both prevention and treatment of TGEV infection in swine.

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“…A pediatric anti-HIV-1 subtype C scFv recombinant phage library was constructed as described previously ( 43 , 44 , 46 , 47 ) with a few modifications. Briefly, one million PBMCs from each of the nine select pediatric cross-neutralizers were pooled and total RNA was isolated by Trizol reagent (Sigma) and then reverse transcribed to cDNA, using the Reverse aid M-MuLV reverse transcriptase (Thermo).…”

Section: Methodsmentioning

confidence: 99%

“…The rescue of phage library was done with M13KO7 helper phage (Stratagene) as mentioned previously ( 44 , 48 ). The phages were then subjected to five rounds of enrichment by bio-panning as described earlier ( 43 ). Briefly, RSC3 core protein was coupled to magnetic beads (MyOne Tosyl activated Dynalbeads, Invitrogen) according to the manufacturer’s instructions, and the antigenic integrity of protein after bead coupling was verified by flow cytometry, using bnAb VRC01.…”

Section: Methodsmentioning

confidence: 99%

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CD4-Binding Site Directed Cross-Neutralizing scFv Monoclonals from HIV-1 Subtype C Infected Indian Children

Kumar

Khan

et al. 2017

Front. Immunol.

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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.

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Cross-neutralizing anti-HIV-1 human single chain variable fragments(scFvs) against CD4 binding site and N332 glycan identified from a recombinant phage library

Cited by 19 publications

References 67 publications

Viral Characteristics Associated with Maintenance of Elite Neutralizing Activity in Chronically HIV-1 Clade C-Infected Monozygotic Pediatric Twins

Viral Characteristics Associated with Maintenance of Elite Neutralizing Activity in Chronically HIV-1 Clade C-Infected Monozygotic Pediatric Twins

Construction and characterization of porcine single-chain fragment variable antibodies that neutralize transmissible gastroenteritis virus in vitro

CD4-Binding Site Directed Cross-Neutralizing scFv Monoclonals from HIV-1 Subtype C Infected Indian Children

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