Design of an Escherichia coli Expressed HIV-1 gp120 Fragment Immunogen That Binds to b12 and Induces Broad and Potent Neutralizing Antibodies

Bhattacharyya, Sanchari; Singh, Pranveer; Rathore, Ujjwal; Purwar, Mansi; Wagner, Denise; Arendt, Heather; DeStefano, Joanne; LaBranche, Celia C.; Montefiori, David C.; Phogat, Sanjay; Varadarajan, Raghavan

doi:10.1074/jbc.m112.425959

Cited by 27 publications

(36 citation statements)

References 62 publications

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“…The neutralization titers are 2-3-fold lower than at week 20. To confirm whether the observed neutralization was Env directed, sera were adsorbed onto immobilized gp120 or trimeric cyclic permutant protein essentially as described previously (34,47). The data ( Table 2) clearly show that the observed neutralization is gp120 directed and could be adsorbed out by incubation with either immobilized gp120 or immobilized trimeric cyclic permutant.…”

Section: Eliciting Broadly Neutralizing Antibodies Against Hiv-1mentioning

confidence: 99%

“…FACS was used to check the antigenic integrity of the bound protein as reported (34,47). 500 l of pooled terminal bleed sera (diluted 1:5 in DMEM) were incubated with 12.5 mg of JRFL gp120-coupled magnetic beads for 2 h at room temperature with gentle rocking.…”

Section: Methodsmentioning

confidence: 99%

“…To screen binding of the CD4 binding site-stabilized outer domain immunogens (b122a, ODEC) (34,49), 96-well plates were coated overnight at 4°C with 25 l of PBS containing 300 ng of protein per well. The wells were washed four times with PBS containing 0.025% Tween 20 (PBST) and blocked with 3% BSA at room temperature for 1 h. Serial dilutions of pooled antisera of each group of animals from week 20 were added to antigen-coated wells, incubated for 2 h at room temperature, and washed 4 times with PBST.…”

Section: Methodsmentioning

confidence: 99%

“…6A). To further validate these results we tested the binding of sera from all four groups to fragment immunogens of the gp120 outer domain that harbor most of the CD4 binding site of gp120 (34,49). Antibodies induced by cyclically permuted gp120 trimers showed substantially higher affinity to these fragment immunogens, indicating the presence of a substantial fraction of CD4bs-specific antibodies (Fig.…”

Section: Eliciting Broadly Neutralizing Antibodies Against Hiv-1mentioning

confidence: 99%

“…In a prior study (34) we showed that sera capable of neutralizing multiple Tier 2 isolates could be elicited by priming with a fragment containing a part of the epitope for the bNAb b12 and boosting with full-length gp120. However, this study employed a year long immunization schedule.…”

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confidence: 99%

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Structure-based Design of Cyclically Permuted HIV-1 gp120 Trimers That Elicit Neutralizing Antibodies

Kesavardhana

Das

Citron

et al. 2017

Journal of Biological Chemistry

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A major goal for HIV-1 vaccine development is an ability to elicit strong and durable broadly neutralizing antibody (bNAb) responses. The trimeric envelope glycoprotein (Env) spikes on HIV-1 are known to contain multiple epitopes that are susceptible to bNAbs isolated from infected individuals. Nonetheless, all trimeric and monomeric Env immunogens designed to date have failed to elicit such antibodies. We report the structure-guided design of HIV-1 cyclically permuted gp120 that forms homogeneous, stable trimers, and displays enhanced binding to multiple bNAbs, including VRC01, VRC03, VRC-PG04, PGT128, and the quaternary epitope-specific bNAbs PGT145 and PGDM1400. Constructs that were cyclically permuted in the V1 loop region and contained an N-terminal trimerization domain to stabilize V1V2-mediated quaternary interactions, showed the highest homogeneity and the best antigenic characteristics. In guinea pigs, a DNA prime-protein boost regimen with these new gp120 trimer immunogens elicited potent neutralizing antibody responses against highly sensitive Tier 1A isolates and weaker neutralizing antibody responses with an average titer of about 115 against a panel of heterologous Tier 2 isolates. A modest fraction of the Tier 2 virus neutralizing activity appeared to target the CD4 binding site on gp120. These results suggest that cyclically permuted HIV-1 gp120 trimers represent a viable platform in which further modifications may be made to eventually achieve protective bNAb responses.The HIV-1 Env displayed on the virion surface mediates entry of the virion into target CD4 ϩ T cells to establish infection. Due to its surface accessibility, Env is the predominant target for neutralizing antibody responses (1, 2). Env is synthesized as a gp160 precursor protein, which is further cleaved into surface proximal gp120 and membrane anchored gp41 subunits. A functional HIV-1 Env spike consists of a trimer of gp120 and gp41 heterodimers. The base of the trimer is proximal to the viral membrane (3) and is held together by trimerization motifs within the N-heptad repeat of gp41 (4 -6). The trimer apex is stabilized by interactions between the V1V2 loops of adjacent gp120 monomers (3). Binding of Env to the CD4 receptor on T cells causes extensive conformational changes in the Env trimer and leads to the formation of an open CD4-bound conformation in which the cryptic, co-receptor binding site becomes accessible. This facilitates interaction of the co-receptor (CCR5 or CXCR4) with Env, further driving the fusion of viral and host cell membranes (3, 7).In natural HIV-1 infection, most of the B cell response is elicited against the Env protein, due to its location on the virion surface and relatively high immunogenicity. The antibody response induced during natural HIV-1 infection is predominantly non-neutralizing and strain specific, due to the shed gp120 (immunodominant decoys) and various other immune evasive mechanisms (8). However, ϳ20% of HIV-1-infected patients develop bNAbs during the course of 1-3 years of infectio...

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Section: Eliciting Broadly Neutralizing Antibodies Against Hiv-1mentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Eliciting Broadly Neutralizing Antibodies Against Hiv-1mentioning

confidence: 99%

mentioning

confidence: 99%

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Structure-based Design of Cyclically Permuted HIV-1 gp120 Trimers That Elicit Neutralizing Antibodies

Kesavardhana

Das

Citron

et al. 2017

Journal of Biological Chemistry

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QTY code designed antibodies for aggregation prevention: A structural bioinformatic and computational study

Li,

Wang,

Tao

et al. 2023

Proteins

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Therapeutic monoclonal antibodies are the most rapidly growing class of molecular medicine, and they are beneficial to the treatment of a broad spectrum of human diseases. However, the aggregation of antibodies during the process of manufacture, distribution, and storage poses significant challenges, potentially compromising efficacy and inducing adverse immune responses. We previously conceived a QTY (glutamine, threonine, tyrosine) code, a simple tool for enhancing protein water‐solubility by systematically pairwise replacing hydrophobic residues L (leucine), V (valine)/I (isoleucine), and F (phenylalanine). The QTY code offers a promising alternative to traditional methods of controlling aggregation in integral transmembrane proteins. In this study, we designed variants of four antibodies applying the QTY code, changing only the β‐sheets. Through the structure‐based aggregation analysis, we found that these QTY antibody variants demonstrated significantly decreased aggregation propensity compared to their wild‐type counter parts. Our results of molecular dynamics simulations showed that the design by QTY code is capable of maintaining the antigen‐binding affinity and structural stability. Our structural informatic and computational study suggests that the QTY code offers a significant potential in mitigating antibody aggregation.

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Influenza Hemagglutinin Head Domain Mimicry by Rational Design

2020

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Despite diligent vaccination efforts, influenza virus infection remains a major cause for respiratory-related illness across the globe. The less-than-optimal immunity conferred by the currently prescribed seasonal vaccines and protracted production times warrant the development of novel vaccines. Induction of an epitope-focused antibody response targeting known neutralization epitopes is a viable strategy to enhance the breadth of protection against rapidly evolving infectious viruses. We report the development of a design framework to mimic the hemagglutinin (HA) head fragment of H1-subtype viruses by delineating the interaction network of invariant residues lining the receptor binding site (RBS); a site targeted by crossreactive neutralizing antibodies. The incorporation of multiple sequence alignment information in our algorithm to fix the construct termini and engineer rational mutations facilitates the facile extension of the design to heterologous (subtypespecific) influenza strains. We evaluated our design protocol by generating head fragments from divergent influenza A H1N1 A/Puerto Rico/8/34 and pH1N1 A/California/07/2009 strains that share a sequence identity of only 74.4% within the HA1 subunit. The designed immunogens exhibited characteristics of a well-ordered protein, and bound conformation-specific RBS targeting antibodies with high affinity, a desirable feature for putative vaccine candidates. Additionally, the bacterial expression of these immunogens provides a low-cost, rapidly scalable alternative.

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Design of an Escherichia coli Expressed HIV-1 gp120 Fragment Immunogen That Binds to b12 and Induces Broad and Potent Neutralizing Antibodies

Cited by 27 publications

References 62 publications

Structure-based Design of Cyclically Permuted HIV-1 gp120 Trimers That Elicit Neutralizing Antibodies

Structure-based Design of Cyclically Permuted HIV-1 gp120 Trimers That Elicit Neutralizing Antibodies

QTY code designed antibodies for aggregation prevention: A structural bioinformatic and computational study

Influenza Hemagglutinin Head Domain Mimicry by Rational Design

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