Purification of recombinant vaccinia virus-expressed monomeric HIV-1 gp120 to apparent homogeneity

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dThe HIV-1 envelope glycoprotein (Env) mediates viral entry into host cells and is the sole target of neutralizing antibodies. Much of the sequence diversity in the HIV-1 genome is concentrated within Env, particularly within its gp120 surface subunit. While dramatic functional diversity exists among HIV-1 Env isolates-observable even in the context of monomeric gp120 proteins as differences in antigenicity and immunogenicity-we have little understanding of the structural features that distinguish Env isolates and lead to isolate-specific functional differences, as crystal structures of truncated gp120 "core" proteins from diverse isolates reveal a high level of structural conservation. Because gp120 proteins are used as prospective vaccine immunogens, it is critical to understand the structural factors that influence their reactivity with antibodies. Here, we studied four full-length, glycosylated gp120 monomers from diverse HIV-1 isolates by using small-angle X-ray scattering (SAXS) to probe the overall subunit morphology and hydrogen/deuterium-exchange with mass spectrometry (HDX-MS) to characterize the local structural order of each gp120. We observed that while the overall subunit architecture was similar among isolates by SAXS, dramatic isolate-specific differences in the conformational stability of gp120 were evident by HDX-MS. These differences persisted even with the CD4 receptor bound. Furthermore, surface plasmon resonance (SPR) and enzyme-linked immunosorbance assays (ELISAs) showed that disorder was associated with poorer recognition by antibodies targeting conserved conformational epitopes. These data provide additional insight into the structural determinants of gp120 antigenicity and suggest that conformational dynamics should be considered in the selection and design of optimized Env immunogens.

Section: Methodsmentioning

confidence: 99%

Isolate-Specific Differences in the Conformational Dynamics and Antigenicity of HIV-1 gp120

Davenport

Guttman

Guo

et al. 2013

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“…Virus stocks that expressed gp160 were subsequently concentrated by sucrose density sedimentation as previously described (30,32). Subunit gp120s were purified from BSC40 cells infected with gp120-expressing recombinant vaccinia viruses as previously described (33).…”

Section: Recombinant Vaccinia Virus (Rvv)mentioning

confidence: 99%

“…Expression of the transgene, under the control of a synthetic early-late promoter of VV, was verified by Western blotting as previously described (33,34). Briefly, monolayers of BSC40 cells were grown to confluence and subsequently infected with recombinant or v-NY parental virus at a multiplicity of infection of 3.…”

Section: Recombinant Vaccinia Virus (Rvv)mentioning

confidence: 99%

Induction of Heterologous Tier 2 HIV-1-Neutralizing and Cross-Reactive V1/V2-Specific Antibodies in Rabbits by Prime-Boost Immunization

Townsley

Mohamed²,

Guo

et al. 2016

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Poxvirus prime-protein boost used in the RV144 trial remains the only immunization strategy shown to elicit a modest level of protection against HIV-1 acquisition in humans. Although neutralizing antibodies (NAb) were generated, they were against sensitive viruses, not the more resistant "tier 2" isolates that dominate circulating strains. Instead, risk reduction correlated with antibodies recognizing epitopes in the V1/V2 region of HIV-1 envelope glycoprotein (Env). Here, we examined whether tier 2 virus NAb and V1/V2-specific non-NAb could be elicited by a poxvirus prime-gp120 boost strategy in a rabbit model. We studied two clade B Envs that differ in multiple parameters, including tissue origin, neutralization sensitivity, and presence of the N197 (N7) glycan that was previously shown to modulate the exposure of conserved epitopes on Env. We demonstrate that immunized rabbits generated cross-reactive neutralizing activities against >50% of the tier 2 global HIV-1 isolates tested. Some of these activities were directed against the CD4 binding site (CD4bs). These rabbits also generated antibodies that recognized protein scaffolds bearing V1/V2 sequences from diverse HIV-1 isolates and mediated antibody-dependent cellular cytotoxicity. However, there are subtle differences in the specificities and the response rates of V1/V2-specific antibodies between animals immunized with different Envs, with or without the N7 glycan. These findings demonstrate that antibody responses that have been correlated with protection against HIV-1 acquisition in humans can be elicited in a preclinical model by a poxvirus prime-gp120 boost strategy and that improvements may be achievable by optimizing the nature of the priming and boosting immunogens. IMPORTANCEThe only vaccine approach shown to elicit any protective efficacy against HIV-1 acquisition is based on a poxvirus prime-protein boost regimen (RV144 Thai trial). Reduction of risk was associated with nonneutralizing antibodies targeting the V1/V2 loops of the envelope protein gp120. However, the modest efficacy (31.2%) achieved in this trial highlights the need to examine approaches and factors that may improve vaccine-induced responses, including cross-reactive neutralizing activities. We show here that rabbits immunized with a novel recombinant vaccinia virus prime-gp120 protein boost regimen generated antibodies that recognize protein scaffolds bearing V1/V2 sequences from diverse HIV-1 isolates and mediated antibody-dependent cellular cytotoxicity. Importantly, immunized rabbits also showed neutralizing activities against heterologous tier 2 HIV-1 isolates. These findings may inform the design of prime-boost immunization approaches and help improve the protective efficacy of candidate HIV-1 vaccines.

“…Env capture assays using D7324 were carried out as previously described (43,60), with slight modifications. In brief, 96-well plates were coated with 10 g/ml of sheep anti-gp120 capture antibody D7324 (Aalto Bio Reagents Ltd., Dublin, Ireland) overnight at room temperature, blocked with phosphate-buffered saline (PBS)-0.05% Tween-5% milk, washed with PBS-0.05% Tween, and incubated for 2 h at 37°C with wild-type (WT) or N7 mutant Env from one of three sources: gp120 generated by recombinant vaccinia virus-infected cells (61), detergent-lysed pseudovirus, or whole pseudovirus. SF162 or JR-FL Env captured on plates was washed before monoclonal antibody (MAb) 697-30D (at 30 g/ml or 3 g/ml, respectively) was added and incubated for 1.5 h at 37°C.…”

mentioning

confidence: 99%

Conserved Role of an N-Linked Glycan on the Surface Antigen of Human Immunodeficiency Virus Type 1 Modulating Virus Sensitivity to Broadly Neutralizing Antibodies against the Receptor and Coreceptor Binding Sites

Townsley

Kozyrev

et al. 2016

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HIV-1 establishes persistent infection in part due to its ability to evade host immune responses. Occlusion by glycans contributes to masking conserved sites that are targets for some broadly neutralizing antibodies (bNAbs). Previous work has shown that removal of a highly conserved potential N-linked glycan (PNLG) site at amino acid residue 197 (N7) on the surface antigen gp120 of HIV-1 increases neutralization sensitivity of the mutant virus to CD4 binding site (CD4bs)-directed antibodies compared to its wild-type (WT) counterpart. However, it is not clear if the role of the N7 glycan is conserved among diverse HIV-1 isolates and if other glycans in the conserved regions of HIV-1 Env display similar functions. In this work, we examined the role of PNLGs in the conserved region of HIV-1 Env, particularly the role of the N7 glycan in a panel of HIV-1 strains representing different clades, tissue origins, coreceptor usages, and neutralization sensitivities. We demonstrate that the absence of the N7 glycan increases the sensitivity of diverse HIV-1 isolates to CD4bs-and V3 loop-directed antibodies, indicating that the N7 glycan plays a conserved role masking these conserved epitopes. However, the effect of the N7 glycan on virus sensitivity to neutralizing antibodies directed against the V2 loop epitope is isolate dependent. These findings indicate that the N7 glycan plays an important and conserved role modulating the structure, stability, or accessibility of bNAb epitopes in the CD4bs and coreceptor binding region, thus representing a potential target for the design of immunogens and therapeutics. A lthough the role of neutralizing antibodies has yet to be determined in the only clinical trial of human immunodeficiency virus type 1 (HIV-1) vaccines that has shown a modest degree of protection, it is generally believed that it would be advantageous for a vaccine to elicit broadly neutralizing antibodies (bNAbs) against diverse primary isolates. Passive administration of neutralizing monoclonal antibodies (MAbs) or bNAbs derived from HIV-1-infected patients has been shown to protect macaques from simian-human immunodeficiency virus (SHIV) infection (1-5). A fraction of HIV-1-infected individuals (ϳ20 to 30%) generate bNAbs within 2 to 4 years of initial infection (6-10). However, generation of bNAbs by active immunization has been a challenge, as no HIV-1 vaccine candidate has successfully elicited antibodies with a similar neutralizing breadth (8, 11).Nevertheless, broadly neutralizing monoclonal antibodies isolated from selected individuals have helped define the targets of bNAbs. These bNAbs are directed against one of five conserved epitopes on HIV-1 envelope glycoprotein (Env); the CD4 binding site (CD4bs), the membrane-proximal ectodomain region (MPER), carbohydrates on the outer domain, a quaternary structure in the V1 and V2 loops, and a newly described epitope present only in cleaved envelope trimers (7,(11)(12)(13). However, HIV-1 has evolved many protective mechanisms to evade host immune respon...