Differential binding of neutralizing and non-neutralizing antibodies to native-like soluble HIV-1 Env trimers, uncleaved Env proteins, and monomeric subunits

Background: The trimeric envelope glycoproteins (Env) on the surface of HIV-1 virions are the targets for neutralizing antibodies (NAbs). No candidate HIV-1 immunogen has yet induced potent, broadly active NAbs (bNAbs). Part of the explanation may be that previously tested Env proteins inadequately mimic the functional, native Env complex. Trimerization and the proteolytic processing of Env precursors into gp120 and gp41 profoundly alter antigenicity, but soluble cleaved trimers are too unstable to serve as immunogens. By introducing stabilizing mutations (SOSIP), we constructed soluble, cleaved Env trimers derived from the HIV-1 subtype A isolate BG505 that resemble native Env spikes on virions both structurally and antigenically. Results: We used surface plasmon resonance (SPR) to quantify antibody binding to different forms of BG505 Env: the proteolytically cleaved SOSIP.664 trimers, cleaved gp120-gp41ECTO protomers, and gp120 monomers. Non-NAbs to the CD4-binding site bound only marginally to the trimers but equally well to gp120-gp41ECTO protomers and gp120 monomers, whereas the bNAb VRC01, directed to the CD4bs, bound to all three forms. In contrast, bNAbs to V1V2 glycan-dependent epitopes bound preferentially (PG9 and PG16) or exclusively (PGT145) to trimers. We also explored the antigenic consequences of three different features of SOSIP.664 gp140 trimers: the engineered inter-subunit disulfide bond, the trimer-stabilizing I559P change in gp41ECTO, and proteolytic cleavage at the gp120-gp41ECTO junction. Each of these three features incrementally promoted native-like trimer antigenicity. We compared Fab and IgG versions of bNAbs and validated a bivalent model of IgG binding. The NAbs showed widely divergent binding kinetics and degrees of binding to native-like BG505 SOSIP.664. High off-rate constants and low stoichiometric estimates of NAb binding were associated with large amounts of residual infectivity after NAb neutralization of the corresponding BG505.T332N pseudovirus. Conclusions: The antigenicity and structural integrity of cleaved BG505 SOSIP.664 trimers render these proteins good mimics of functional Env spikes on virions. In contrast, uncleaved gp140s antigenically resemble individual gp120-gp41ECTO protomers and gp120 monomers, but not native trimers. Although NAb binding to functional trimers may thus be both necessary and sufficient for neutralization, the kinetics and stoichiometry of the interaction influence the neutralizing efficacy of individual NAbs

Gp120/CD4 blocking antibodies are frequently elicited in ART-naïve chronically HIV-1 infected individuals

Antibodies with the ability to block the interaction of HIV-1 envelope glycoprotein (Env) gp120 with CD4, including those overlapping the CD4 binding site (CD4bs antibodies), can protect from infection by HIV-1, and their elicitation may be an interesting goal for any vaccination strategy. To identify gp120/CD4 blocking antibodies in plasma samples from HIV-1 infected individuals we have developed a competitive flow cytometry-based functional assay. In a cohort of treatment-naïve chronically infected patients, we showed that gp120/CD4 blocking antibodies were frequently elicited (detected in 97% plasma samples) and correlated with binding to trimeric HIV-1 envelope glycoproteins. However, no correlation was observed between functional CD4 binding blockade data and titer of CD4bs antibodies determined by ELISA using resurfaced gp120 proteins. Consistently, plasma samples lacking CD4bs antibodies were able to block the interaction between gp120 and its receptor, indicating that antibodies recognizing other epitopes, such as PGT126 and PG16, can also play the same role. Antibodies blocking CD4 binding increased over time and correlated positively with the capacity of plasma samples to neutralize the laboratory-adapted NL4.3 and BaL virus isolates, suggesting their potential contribution to the neutralizing workforce of plasma in vivo. Determining whether this response can be boosted to achieve broadly neutralizing antibodies may provide valuable information for the design of new strategies aimed to improve the anti-HIV-1 humoral response and to develop a successful HIV-1 vaccine

Conjugation of a Toll-Like Receptor Agonist to Glycans of an HIV Native-Like Envelope Trimer Preserves Neutralization Epitopes

Small molecule adjuvants are attractive for enhancing broad protection and durability of immune responses elicited by subunit vaccines. Covalent attachment of an adjuvant to an immunogen is particularly attractive because it simultaneously delivers both entities to antigen presenting cells resulting in more efficient immune activation. There is, however, a lack of methods to conjugate small molecule immune potentiators to viral glycoprotein immunogens without compromising epitope integrity. We describe herein a one-step enzymatic conjugation approach for the covalent attachment of small molecule adjuvants to N-linked glycans of viral glycoproteins. It involves the attachment of an immune potentiator to CMP-Neu5AcN 3 by Cu(I)-catalyzed azide-alkyne 1,3-cycloaddition followed by sialyltransferase-mediated transfer to N-glycans of a viral glycoprotein. The method was employed to modify a native-like HIV envelope trimer with a Toll-like receptor 7/8 agonist. The modification did not compromise Env-trimer recognition by several broadly neutralization antibodies. Electron microscopy confirmed structural integrity of the modified immunogen

HIV-1 superinfection results in broad polyclonal neutralizing antibodies

<div><p>HIV-1 vaccines designed to date have failed to elicit neutralizing antibodies (Nabs) that are capable of protecting against globally diverse HIV-1 subtypes. One relevant setting to study the development of a strong, cross-reactive Nab response is HIV-1 superinfection (SI), defined as sequential infections from different source partners. SI has previously been shown to lead to a broader and more potent Nab response when compared to single infection, but it is unclear whether SI also impacts epitope specificity and if the epitopes targeted after SI differ from those targeted after single infection. Here the post-SI Nab responses were examined from 21 Kenyan women collectively exposed to subtypes A, C, and D and superinfected after a median time of ~1.07 years following initial infection. Plasma samples chosen for analysis were collected at a median time point ~2.72 years post-SI. Because previous studies of singly infected populations with broad and potent Nab responses have shown that the majority of their neutralizing activity can be mapped to 4 main epitopes on the HIV-1 Envelope, we focused on these targets, which include the CD4-binding site, a V1/V2 glycan, the N332 supersite in V3, and the membrane proximal external region of gp41. Using standard epitope mapping techniques that were applied to the previous cohorts, the present study demonstrates that SI did not induce a dominant Nab response to any one of these epitopes in the 21 women. Computational sera delineation analyses also suggested that 20 of the 21 superinfected women’s Nab responses could not be ascribed a single specificity with high confidence. These data are consistent with a model in which SI with diverse subtypes promotes the development of a broad polyclonal Nab response, and thus would provide support for vaccine designs using multivalent HIV immunogens to elicit a diverse repertoire of Nabs.</p></div

Structure-guided envelope trimer design in HIV-1 vaccine development: a narrative review

Introduction: The development of a human immunodeficiency virus 1 (HIV-1) vaccine remains a formidable challenge. An effective vaccine likely requires the induction of broadly neutralizing antibodies (bNAbs), which likely involves the use of native-like HIV-1 envelope (Env) trimers at some or all stages of vaccination. Development of such trimers has been very difficult, but much progress has been made in the past decade, starting with the BG505 SOSIP trimer, elucidation of its atomic structure and implementing subsequent design iterations. This progress facilitated understanding the weaknesses of the Env trimer, fuelled structure-guided HIV-1 vaccine design and assisted in the development of new vaccine designs. This review summarizes the relevant literature focusing on studies using structural biology to reveal and define HIV-1 Env sites of vulnerability; to improve Env trimers, by creating more stable versions; understanding antibody responses in preclinical vaccination studies at the atomic level; understanding the glycan shield; and to improve “on-target” antibody responses versus “off-target” responses. Methods: The authors conducted a narrative review of recently published articles that made a major contribution to HIV-1 structural biology and vaccine design efforts between the years 2000 and 2021. Discussion: The field of structural biology is evolving at an unprecedented pace, where cryo-electron microscopy (cryo-EM) and X-ray crystallography provide complementary information. Resolving protein structures is necessary for defining which Env surfaces are accessible for the immune system and can be targeted by neutralizing antibodies. Recently developed techniques, such as electron microscopy-based polyclonal epitope mapping (EMPEM) are revolutionizing the way we are analysing immune responses and shed light on the immunodominant targets on new vaccine immunogens. Such information accelerates iterative vaccine design; for example, by reducing undesirable off-target responses, while improving immunogens to drive the more desirable on-target responses. Conclusions: Resolving high-resolution structures of the HIV-1 Env trimer was instrumental in understanding and improving recombinant HIV-1 Env trimers that mimic the structure of viral HIV-1 Env spikes. Newly emerging techniques in structural biology are aiding vaccine design efforts and improving immunogens. The role of structural biology in HIV-1 vaccine design has indeed become very prominent and is unlikely to diminish any time soon

Interplay between viral Tat protein and c-Jun transcription factor in controlling LTR promoter activity in different human immunodeficiency virus type I subtypes

HIV-1 transcription depends on cellular transcription factors that bind to sequences in the long-terminal repeat (LTR) promoter. Each HIV-1 subtype has a specific LTR promoter configuration, and minor sequence changes in transcription factor binding sites (TFBSs) or their arrangement can influence transcriptional activity, virus replication and latency properties. Previously, we investigated the proviral latency properties of different HIV-1 subtypes in the SupT1 T cell line. Here, subtype-specific latency and replication properties were studied in primary PHA-activated T lymphocytes. No major differences in latency and replication capacity were measured among the HIV-1 subtypes. Subtype B and AE LTRs were studied in more detail with regard to a putative AP-1 binding site using luciferase reporter constructs. c-Jun, a member of the AP-1 transcription factor family, can activate both subtype B and AE LTRs, but the latter showed a stronger response, reflecting a closer match with the consensus AP-1 binding site. c-Jun overexpression enhanced Tat-mediated transcription of the viral LTR, but in the absence of Tat inhibited basal promoter activity. Thus, c-Jun can exert a positive or negative effect via the AP-1 binding site in the HIV-1 LTR promoter, depending on the presence or absence of Ta

On-Chip Detector Cooling for Space Applications

Vibration-free miniature Joule-Thomson (JT) coolers are of interest for cooling small optical detectors for future space missions. The European Space Agency is interested in on-chip detector cooling for the temperature range 70¶K¶-¶250¶K. This is the topic of a new project at the University of Twente. In this project, the focus is on the integration of Micro-Electro-Mechanical Systems (MEMS) based JT coolers with small detectors for space applications. Furthermore, distributed micro cooling will be investigated, which means that multiple cold tips will be driven by a single compressor. The progress of this project is discussed in this paper. The application of JT coolers in the temperature range 70¶K¶-¶250¶K is investigated. The working fluid in a JT cold stage is optimized on basis of the thermodynamical properties of the fluid. The results of this optimization are discussed. Furthermore, a static model of the counter flow heat exchanger is built, in order to simulate the performance of a micro JT cold stage. The results of the simulations are compared with experimental data

Conjugation of a Toll-Like Receptor Agonist to Glycans of an HIV Native-Like Envelope Trimer Preserves Neutralization Epitopes

Small molecule adjuvants are attractive for enhancing broad protection and durability of immune responses elicited by subunit vaccines. Covalent attachment of an adjuvant to an immunogen is particularly attractive because it simultaneously delivers both entities to antigen presenting cells resulting in more efficient immune activation. There is, however, a lack of methods to conjugate small molecule immune potentiators to viral glycoprotein immunogens without compromising epitope integrity. We describe herein a one-step enzymatic conjugation approach for the covalent attachment of small molecule adjuvants to N-linked glycans of viral glycoproteins. It involves the attachment of an immune potentiator to CMP-Neu5AcN3 by Cu(I)-catalyzed azide-alkyne 1,3-cycloaddition followed by sialyltransferase-mediated transfer to N-glycans of a viral glycoprotein. The method was employed to modify a native-like HIV envelope trimer with a Toll-like receptor 7/8 agonist. The modification did not compromise Env-trimer recognition by several broadly neutralization antibodies. Electron microscopy confirmed structural integrity of the modified immunogen

Characteristic of ART-naive HIV-1 infected patients.

<p>Characteristic of ART-naive HIV-1 infected patients.</p