[Show abstract][Hide abstract] ABSTRACT: 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 crossneutralization 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.
PLoS ONE 05/2015; 10(5):e0125575. DOI:10.1371/journal.pone.0125575 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Little is known about the neutralizing antibodies induced in HIV-1 patients on antiretroviral treatment, which constitute an interesting group of individuals with improved B cell profile. Plasma samples from 34 HIV-1 seropositive antiretroviral drug treated (ART) patients were tested for neutralization against a panel of 14 subtype-A, B and C tier 1 and tier 2 viruses in TZM-bl assay. Of the 34 plasma samples, remarkably all the plasma samples were able to neutralize at least one virus while 32 (94 %) were found to neutralize ≥50 % viruses tested. In terms of overall neutralization frequency, approximately 86 %, 68 % and 17 % of the virus/plasma combinations showed 50 % neutralizing activity at 1 > 60, 1 ≥ 200 and 1 ≥ 2000 dilutions respectively. The improvement in neutralizing activity was shown to be associated with ART in two follow up patients. The neutralization of viruses by two representative plasma samples, AIIMS221 and AIIMS265, was exclusively mediated by immunoglobulin G fractions independent of ART drugs and IgG retained cross-reactive binding to recombinant gp120 proteins. We observed a positive trend of neutralization with duration of ART (p = 0.06), however no such correlation was found with clinical and immunological variables like CD4 count (p = 0.35), viral load (p = 0.09) and plasma total IgG (p = 0.46). Our study suggests that the plasma antibodies from ART patients display high neutralizing activity most likely due to an improved B cell function induced by ART despite low antigenic stimulation.
[Show abstract][Hide abstract] ABSTRACT: The 24(th) Antibody Engineering & Therapeutics meeting brought together a broad range of participants who were updated on the latest advances in antibody research and development. Organized by IBC Life Sciences, the gathering is the annual meeting of The Antibody Society, which serves as the scientific sponsor. Preconference workshops on 3D modeling and delineation of clonal lineages were featured, and the conference included sessions on a wide variety of topics relevant to researchers, including systems biology; antibody deep sequencing and repertoires; the effects of antibody gene variation and usage on antibody response; directed evolution; knowledge-based design; antibodies in a complex environment; polyreactive antibodies and polyspecificity; the interface between antibody therapy and cellular immunity in cancer; antibodies in cardiometabolic medicine; antibody pharmacokinetics, distribution and off-target toxicity; optimizing antibody formats for immunotherapy; polyclonals, oligoclonals and bispecifics; antibody discovery platforms; and antibody-drug conjugates.
[Show abstract][Hide abstract] ABSTRACT: A region in the conserved 5' long terminal repeat (LTR) promoter of the integrated HIV-1C provirus was identified for effective targeting by a short double-stranded RNA (dsRNA) to cause heterochromatization leading to a long-lasting decrease in viral transcription, replication and subsequent productive infection in human host cells.
Small interfering RNAs (siRNAs) were transfected into siHa cells containing integrated LTR-luciferase reporter constructs and screened for efficiency of inducing transcriptional gene silencing (TGS). TGS was assessed by a dual luciferase assay and real-time PCR. Chromatin modification at the targeted region was also studied. The efficacy of potent siRNA was then checked for effectiveness in TZM-bl cells and human peripheral blood mononuclear cells (PBMCs) infected with HIV-1C virus. Viral Gag-p24 antigen levels were determined by ELISA.
One HIV-1C LTR-specific siRNA significantly decreased luciferase activity and its mRNA expression with no such effect on HIV-1B LTR. This siRNA-mediated TGS was induced by histone methylation, which leads to heterochromatization of the targeted LTR region. The same siRNA also substantially suppressed viral replication in TZM-bl cells and human PBMCs infected with various HIV-1C clinical isolates for ≥3 weeks after a single transfection, even of a strain that had a mismatch in the target region.
We have identified a potent dsRNA that causes long-term suppression of HIV-1C virus production in vitro and ex vivo by heritable epigenetic modification at the targeted C-LTR region. This dsRNA has promising therapeutic potential in HIV-1C infection, the clade responsible for more than half of AIDS cases worldwide.
[Show abstract][Hide abstract] ABSTRACT: One approach to the development of an HIV vaccine is to design a protein template which can present gp120 epitopes inducing cross-neutralizing antibodies. To select a V3 sequence for immunogen design, we compared the neutralizing activities of 18 anti-V3 monoclonal antibodies (mAbs) derived from Cameroonian and Indian individuals infected with clade AG and C, respectively. It was found that V3 mAbs from the Cameroonian patients were significantly more cross-neutralizing than those from India. Interestingly, superior neutralizing activity of Cameroonian mAbs was also observed among the nine VH5-51/VL lambda genes encoding V3 mAbs which mediate a similar mode of recognition. This correlated with higher relative binding affinity to a variety of gp120s and increased mutation rates in V3 mAbs from Cameroon. These results suggest that clade C V3 is probably weakly immunogenic and that the V3 sequence of CRF02_AG viruses can serve as a plausible template for vaccine immunogen design.
[Show abstract][Hide abstract] ABSTRACT: Background
Production 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.
An 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.
This 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.
[Show abstract][Hide abstract] ABSTRACT: Subtypes of human immunodeficiency virus type 1 circulating in 21 north Indian patients were characterized based on the partial sequence of the gp120 envelope protein. A majority of viruses (85.7%, 18/21) were subtype C, while 14.3% (3/21) were subtype A. Sequence analysis revealed that the V3 region was highly conserved compared with V4 and V5. The predicted use of co-receptors indicated exclusive usage of R5, except for two subtype A viruses (AIIMS279 and AIIMS281). Our results demonstrate conservation within the V3 loop of subtype C viruses, and suggest the emergence of non-clade C viruses in the north Indian population.
The Journal of Microbiology 10/2012; 50(5):869-73. DOI:10.1007/s12275-012-2136-z · 1.44 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Highly efficient neutralization of human
immunodeficiency viruses by plasma from
antiretroviral drug treated patients is mediated
by IgG fractions
R Andrabi, M Makhdoomi, R Kumar, M Bala, T Velpandian, K Luthra*
From AIDS Vaccine 2012
Boston, MA, USA. 9-12 September 2012
Little is known about the neutralizing activity in patients
on antiretroviral therapy (ART), as most recent studies
have focused on drug naïve individuals. ART may lead
to a significant increase in B cell numbers and normalization
of B cell subpopulations, providing a possible
explanation for improved B cell responses after ART.
Thirty-four HIV-1 seropositive patients on ART (25 males
and 9 females) within the age range of 20-55 years were
recruited in this study. The patients had a median CD4
count and viral load of 283 cells and 178 RNA copies
respectively, and were on treatment for a few days up to
two years. Heat inactivated plasma samples were tested for
neutralization against a panel of 14 subtype-A, B and C
tier 1 and tier 2 viruses in TZM-bl assay.
Of the 34 plasma samples, remarkably all the plasma
samples were able to neutralize at least one virus while
32 (94%) samples were found to neutralize ≥50% viruses
tested. Clustering analysis revealed that AIIMS253 (a
clade-C virus) was the most sensitive while RHPA4259.7
(a clade-B isolate) was most resistant to antibody neutralization.
The Immunoglobulin-G fractions from two
representative samples AIIMS221 and AIIMS265 were
shown to mediate neutralization exclusively. The IgG
fractions retained binding to subtype-A, B and C recombinant
gp120 proteins. We did not find any association of
mean reciprocal ID50 neutralization titers with the
plasma levels of ART drugs and clinical and immunological
variables like CD4 count (p=0.35), viral load (p=0.37)
and plasma total IgG (p=0.46). However we observed a
positive association of neutralization with duration of
ART (p=0.02) with a similar trend in two follow up
Plasma antibodies from patients on ART display high
neutralizing activity most likely due to an improved B
cell function induced by ART despite low antigenic
[Show abstract][Hide abstract] ABSTRACT: Background
Analysis of human monoclonal antibodies (mAbs) developed from HIV-1 infected donors have enormously contributed to the identification of neutralization sensitive epitopes on the HIV-1 envelope glycoprotein. The third variable region (V3) is a crucial target on gp120, primarily due to its involvement in co-receptor (CXCR4 or CCR5) binding and presence of epitopes recognized by broadly neutralizing antibodies.
Thirty-three HIV-1 seropositive drug naive patients (18 males and 15 females) within the age range of 20–57 years (median = 33 years) were recruited in this study for mAb production. The mAbs were selected from EBV transformed cultures with conformationally constrained Cholera-toxin-B containing V3C (V3C-CTB) fusion protein. We tested the mAbs for their binding with HIV-1 derived proteins and peptides by ELISA and for neutralization against HIV-1 viruses by TZM-bl assays.
We isolated three anti-V3 mAbs, 277, 903 and 904 from the cells of different individuals. The ELISA binding revealed a subtype-C and subtype-A specific binding of antibody 277 and 903 while mAb 904 exhibited cross reactivity also with subtype-B V3. Epitope mapping of mAbs with overlapping V3 peptides showed exclusive binding to V3 crown. The antibodies displayed high and low neutralizing activity against 2/5 tier 1 and 1/6 tier 2 viruses respectively. Overall, we observed a resistance of the tier 2 viruses to neutralization by the anti-V3 mAbs, despite the exposure of the epitopes recognized by these antibodies on two representative native viruses (Du156.12 and JRFL), suggesting that the affinity of mAb might equally be crucial for neutralization, as the epitope recognition.
Our study suggests that the anti-V3 antibodies derived from subtype-C infected Indian patients display neutralization potential against tier 1 viruses while such activity may be limited against more resistant tier 2 viruses. Defining the fine epitope specificities of these mAbs and further experimental manipulations will be helpful in identification of epitopes, unique to clade C or shared with non-clade C viruses, in context of V3 region.
[Show abstract][Hide abstract] ABSTRACT: Purpose/Objective: HIV1-the causative agent of AIDS, is the most
infectious and epidemic virus worldwide. Till now there is no permanent
cure for HIV1 infection and Current therapy reduces viral gene
expression and its infection through inhibition of viral proteins
(proteases, integrase and reverse transcriptase), blockade of gp120 and
CD4 interaction and degradation of RNA, however targeting at the
transcription level of the integrated provirus has been less explored. 5’
LTR promoter of HIV1 regulates the expression of all downstream viral
genes and shows variation in sequences and transcription factor
binding sites in a clade specific manner. Indian AIDS patients are
commonly infected with HIV-1C compared to HIV-1B in developed
countries. In our study, we have targeted the HIV-1C LTR promoter
using dsRNA to induce transcriptional gene silencing (TGS) and decrease
viral gene transcription.
Materials and methods: In this study, SiHa cell line stably expressing
the luciferase reporter under the HIV-1C LTR promoter was used in
the presence of Tat protein. Luciferase expression (mRNA) and activity
was measured by Real-Time PCR and dual luciferase assay respectively.
The chromatin status after dsRNA transfection was studied using
CHART-PCR and histone modifications at the targeted region of LTR
were analyzed by ChIP assay. HIV-1C infected cells were transfected
with siRNA and p24 assay was done at different time intervals after
Results: Multiple siRNAs targeting the U3 region of HIV-1C 5’ LTR
were screened using dual luciferase assay and Real-time PCR. One
siRNA (S4) showed significant decrease in luciferase activity and
mRNA expression 72 h post transfection. The S4 mediated TGS was
caused by heterochromatization as seen by the reduced accessibility of
MNAse in CHART PCR assay. Histone methylation of the targeted
region was checked after siRNA transfection by ChIP assay. Our results
showed that the heterochromatization of the HIV1-C LTR promoter
was mediated by histone methylation. We also observed that S4 siRNA
reduces HIV-1 gene transcription and their productive infection in
HIV-1C infected cells after transfection.
Conclusions: Heterochromatization of HIV-1C 5’ LTR by dsRNA can
suppress downstream viral gene expression and thereby productive
HIV1 infection. Thus dsRNA mediated TGS can be used as a
therapeutic modality for HIV-1C infection in future.
[Show abstract][Hide abstract] ABSTRACT: Broadly cross neutralizing antibodies (NAbs) are generated in a group of HIV-1 infected individuals during the natural infection, but little is known about their prevalence in patients infected with viral subtypes from different geographical regions. We tested here the neutralizing efficiency of plasma antibodies from 80 HIV-1 infected antiretroviral drug naive patients against a panel of subtype-B and C tier 2 viruses. We detected cross-neutralizing antibodies in approximately 19-27% of the plasma, however the subtype-C specific neutralization efficiency predominated (p = 0.004). The neutralizing activity was shown to be exclusively mediated by the immunoglobulin G (IgG) fraction in the representative plasma samples. Epitope mapping of three, the most cross-neutralizing plasma (CNP) AIIMS206, AIIMS239 and AIIMS249 with consensus-C overlapping envelope peptides revealed ten different binding specificities with only V3 and IDR being common. The V3 and IDR were highly antigenic regions but no correlation between their reciprocal Max50 binding titers and neutralization was observed. In addition, the neutralizing activity of CNP was not substantially reduced by V3 and gp41 peptides except a modest contribution of MPER peptide. The MPER was rarely recognized by plasma antibodies though antibody depletion and competition experiments demonstrated MPER dependent neutralization in two out of three CNP. Interestingly, the binding specificity of one of the CNP (AIIMS206) overlapped with broadly neutralizing mAb 2F5 epitope. Overall, the data suggest that, despite the low immunogenicity of HIV-1 MPER, the antibodies directed to this region may serve as crucial reagents for HIV-1 vaccine design.
PLoS ONE 08/2012; 7(8):e43704. DOI:10.1371/journal.pone.0043704 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In this cross-sectional study, we evaluated the efficiency of the plasma of 38 antiretroviral-naïve HIV-1-infected children from northern India against a standard panel of pseudoviruses (3 clade C and 3 clade B) by TZM-bl assay. Neutralization potential was observed to a variable extent, with a potency ranging up to reciprocal ID(50) titers of 1967. Cross-neutralization was observed in 28.9 % (11/38) of the children. There was a significant positive correlation between viremia and neutralization efficiency against two of the viruses studied (Du172 r = 0.49; p = 0.007 and RHPA r = 0.47; p = 0.01), suggesting that persistent antigenic stimulation is necessary for the generation of broadly neutralizing antibody responses in these children. Further mapping of the epitope specificities of the neutralization determinants in the polyclonal plasma would provide important information for immunogen design.
Archives of Virology 06/2012; 157(9):1797-801. DOI:10.1007/s00705-012-1357-0 · 2.39 Impact Factor