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Presentation of HIV-1 envelope glycoprotein trimers on diverse nanoparticle platforms

Presentation of HIV-1 envelope glycoprotein trimers on diverse nanoparticle platforms Downloaded from https://journals.lww.com/co-hivandaids by BhDMf5ePHKav1zEoum1tQfN4a+kJLhEZgbsIHo4XMi0hCywCX1AWnYQp/IlQrHD35y8U/jUqeEyVMweXB0LtB1dl03evjuXlpE8l2qkMB64= on 12/16/2019 REVIEW URRENT Presentation of HIV-1 envelope glycoprotein PINION trimers on diverse nanoparticle platforms a a,b Philip J.M. Brouwer and Rogier W. Sanders Purpose of review We will discuss recent advances in the development of nanoparticle vaccines presenting HIV-1 envelope trimer vaccines and the immunological mechanisms by which they act. Recent findings The multivalent presentation of Env trimers on nanoparticles is a promising strategy to increase Env immunogenicity. Recent studies have shed light on how Env nanoparticles increase lymph node trafficking and germinal center formation by using the lectin-mediated complement pathway and enhancing the interaction with naı¨ve B cells. Meanwhile, research on different nanoparticle platforms has resulted in improved designs, such as liposomes with improved stability, and the emergence of novel platforms such as protein nanoparticles that self-assemble in vitro. Immmunogenicity studies with these nanoparticles delineate the advantages and expose the limitations of the different nanoparticle platforms. Summary It is becoming increasingly clear that HIV-1 vaccine research might benefit greatly from using nanoparticles presenting Env trimers, particularly during the priming stage of immunization. Among the different nanoparticles that are being pursued, in vitro-assembling nanoparticles allow for greater control of Env quality making them a promising nanoparticle platform. Keywords HIV-1 Env trimer, liposome, nanoparticle presentation, self-assembling protein nanoparticle, virus-like particle INTRODUCTION based-design has resulted in the development of native-like Env trimers that have improved stability, With around 2 million new cases of HIV worldwide increased yield and reduced exposure of nonneutr- the need for a vaccine that can prevent infection alizing Abs [4,5,7 – 10]. Despite these improvements, remains as high as ever. To deal with the large native-like Env trimers generally induce NAbs with sequence diversity of HIV-1 it is widely accepted narrow specificity that are relatively short-lived. that a successful vaccine should induce broadly Presenting immunogens in a multivalent way on neutralizing antibodies (bNAbs) [1,2]. These bNAbs nanoparticles is a well established strategy to recognize and bind epitopes that are conserved increase their immunogenicity. Here, we review among a wide range of HIV-1 genotypes. The only the most recent advances in Env nanoparticle design target for (b)NAbs is the envelope glycoprotein and the immunological observations made using (Env), a trimer of heterodimers, consisting of three them. gp120 and three gp41 subunits that interact non- covalently. Over the years various forms of recom- binantly expressed Env have been used in attempts Department of Medical Microbiology, Amsterdam UMC, University of to generate a vaccine that could induce NAbs. An Amsterdam, Amsterdam, the Netherlands and Department of Microbi- important breakthrough was the development of ology and Immunology, Weill Medical College of Cornell University, New native-like trimers exemplified by the prototype York, New York, USA BG505 SOSIP.664 trimer [3]. These soluble and sta- Correspondence to Rogier W. Sanders, Department of Medical Micro- bilized native-like mimics of functional Env present biology, Amsterdam UMC, University of Amsterdam, 1105 AZ, Amster- on infectious virus were the first immunogens to dam, the Netherlands. Tel: +31 20 5667768; fax: +31 20 6916531; e-mail: r.w.sanders@amc.uva.nl elicit potent and consistent NAb responses against neutralization-resistant clinically relevant (Tier-2) Curr Opin HIV AIDS 2019, 14:302–308 viruses in animal models [4 – 6]. Since then, structure DOI:10.1097/COH.0000000000000549 www.co-hivandaids.com Volume 14  Number 4  July 2019 Copyright © 2019 Wolters Kluwer Health, Inc. All rights reserved. Downloaded from https://journals.lww.com/co-hivandaids by BhDMf5ePHKav1zEoum1tQfN4a+kJLhEZgbsIHo4XMi0hCywCX1AWnYQp/IlQrHD35y8U/jUqeEyVMweXB0LtB1dl03evjuXlpE8l2qkMB64= on 12/16/2019 HIV-1 Env trimer nanoparticle immunogens Brouwer and Sanders monomeric antigens [16]. Recently, Tokatlian et al. KEY POINTS provided new insights into the immunological mechanisms behind the improved trafficking to Multivalent presentation of Env trimers on nanoparticles lymph nodes of nanoparticles carrying highly gly- increases germinal center responses by improving the && cosylated antigens such as Env [17 ]. Nanoparticles trafficking to and retention in lymph nodes by a complement-dependent mechanism and by enhancing presenting Env trimers where present in lymph the activation of naı¨ve B cells. node follicles, germinal centers, and on fDCs in much higher quantities than their soluble counter- Improvements in VLP, liposome, and in vivo-assembling parts. This was shown to be largely dependent on protein nanoparticle platforms include enhanced complement, as colocalization of particles on fDCs stability and improved Env quality and valency. A new nanoparticle platform has emerged, which consists of in was significantly reduced in mice lacking comple- vitro-assembling protein nanoparticle. ment component C3 or its receptors Cr1/2. They further showed that mannose-binding lectin bound Although Env nanoparticles were generally able to significantly better to nanoparticles than soluble increase Env-binding titers, the modest and inconsistent increase of NAb titers by various nanoparticle immunogens and that the mannose-binding lectin platforms suggest that other factors (i.e. Env pathway mediated the observed fDC targeting && antigenicity, epitope accessibility) other than multivalent [17 ]. Thus, as a result of increased avidity by mul- presentation are critical for optimal exploitation of tivalent presentation of Env, nanoparticles more nanoparticle platforms. efficiently initiate the mannose-binding lectin – pathway resulting in higher deposition of comple- The ability to purify Env trimers prior to assembly in liposomes and in vitro-assembling protein nanoparticles ment. This subsequently leads to more efficient allows for control of Env quality making them promising trafficking to lymph node follicles and presentation nanoparticle platforms. on fDCs. Once in the lymph nodes, nanoparticles will bind more avidly to B-cell receptors (BCRs) on naive cognate B cells than soluble immunogens, enhanc- IMMUNOLOGICAL MECHANISMS OF ing BCR clustering, intracellular BCR-mediated sig- ACTION OF NANOPARTICLES naling, and subsequent activation of the B cell. The success of the commercially available HPV and Indeed, several studies using Env nanoparticles HBV vaccines and the promising preclinical data for and Env-specific B cells show that nanoparticle influenza, EBV and malaria nanoparticle vaccines, presentation enhances B-cell activation in vitro reinforce the paradigm that multivalent presenta- (Brouwer et al. in revision) [8,18 ,19,20,21]. The tion of an immunogen can enhance its immunoge- ability to more efficiently activate cognate B cells nicity [11 – 14]. In light of this, Env trimers have may be of particular relevance in the context of been presented on various nanoparticle platforms of HIV-1 immunogens as bNAb precursors are gener- which several have shown clear improvements in ally highly infrequent and have low affinity for Env && Env immunogenicity. Meanwhile, the immunolog- [22]. Recently, Abbott et al. [23 ] tested the effects of ical mechanisms of action of nanoparticle vaccines immunogen affinity and avidity on germinal center are being elucidated and the recent findings are formation in mice that had a range of precursor discussed here. The improved immunogenicity of frequencies of a specific germline B cell. At physio- nanoparticle immunogens is rooted in several logical affinities (0.5 mMK ) and frequencies (one in immunological processes including antigen traffick- 1  10 cells), nanoparticles were able to expand ing to lymph nodes, activation of antigen-specific B specific germline B cells whereas with the soluble cells, and uptake by and activation of antigen pre- counterpart these B cells were undetectable. Further- senting cells [15]. It is becoming increasingly clear more, even with 10-fold lower precursor frequencies that the benefit of nanoparticles is a result of the and a 16,000 lower affinity, nanoparticle immu- enhancement of multiple immunological processes nogens outcompeted the monomer with respect to involving both innate and adaptive responses. the quantity of detectable specific B cells 8 days post && To mount an antibody response, an immunogen immunization [23 ]. has to find its way into the draining lymph nodes Inducing antigen-presenting cells to migrate to where it will interact with B cells and follicular lymph nodes is a crucial process in the development dendritic cells (fDCs). In the case of direct lymphatic of a humoral response as they prime cognate follic- drainage, antigens will drain from the administra- ular T helper cells that drive B-cell affinity matura- tion site by interstitial flow towards a lymphatic tion in the germinal centers. Nanoparticles have vessel. As particulate antigens have lower diffusion been shown to increase uptake by and activation rates they will be delivered more effectively than of dendritic cells over soluble antigen, with a 1746-630X Copyright  2019 Wolters Kluwer Health, Inc. All rights reserved. www.co-hivandaids.com 303 Copyright © 2019 Wolters Kluwer Health, Inc. All rights reserved. Broad neutralising and non-neutralising antibodies marked dependency on nanoparticle size [24 – 27]. A common strategy to increase the quantity of Env The improved uptake of particles with a similar size on the VLP surface is truncating the cytoplasmic tail. to that of viruses (20 – 200 nm) has led to specula- Recently, Stano et al. [39] were able to further tions that DCs have evolved to recognize virus-sized increase Env quantity by generating a VLP produc- particles [26,28,29]. However, in the case of recep- tion platform that yielded VLPs with more than 100 tor-mediated endocytosis the higher avidity by Env trimers on the surface. How these higher nanoparticle immunogens may also play a role in valency VLPs perform in vivo remains to be the enhanced uptake [30]. To what extent and by addressed. An interesting approach to improve the which mechanism Env nanoparticles increase anti- immunogenicity of Env VLPs was introduced by gen presentation remains to be studied. Elsayed et al. [40], who generated Env VLPs that contained helper T-cell epitopes of tetanus toxoid. Pre-immunization of mice with tetanus toxoid led DIVERSE NANOPARTICLE PLATFORMS to significantly increased Env-specific IgG titers, FOR HIV-1 ENV TRIMER PRESENTATION which was shown to be caused by the intrastructural The immunological arguments presented above pro- help conferred by the helper T-cell epitope(s) [40]. vide a sound rationale for presenting Env trimers on The elegance of this approach lies in the fact that nanoparticles. Although considerable progress has humans have been vaccinated against tetanus dur- been made, several problems, including poor in vivo ing childhood and that the tetanus memory T cells stability of Env-nanoparticles, difficulties in ensuring can be harnessed to help HIV-1 Env immunogenic- and preserving native-like Env structure, and produc- ity, at least in theory. tion issues have limited the improvements in Env Despite these recent advances, producing Env immunogenicity conferred by nanoparticle presen- VLPs to yields that are sufficient for testing in larger tation so far. Below, we discuss the main nanoparticle animal models (rabbits, macaques) remains a critical platforms that are currently being explored. bottleneck. Although the yield of Env-VLPs could be increased by optimizing plasmid combinations, it was estimated that per unit time, more than 100- Virus-like particles fold less doses could be produced compared to SOSIP One of the earliest nanoparticles that have been trimers [38]. Combining the described strategies explored are virus-like particles (VLPs). These multi- that enhance trimeric integrity and quantity of protein, enveloped particles are produced by presented Env and finding ways to increase VLP cotransfecting the gag and env gene, resulting in expression will probably be crucial to solve the the formation of replication-incompetent particles multiple limitations Env VLPs have. that mimic the shape and conformation of the HIV- 1 virus. However, HIV-1 particles generally only Liposomes present on average 7 – 14 Env molecules per particle of which a substantial fraction is nonfunctional Liposomes are unilamellar or multilamellar vesicles, with a nonnative structure, including uncleaved which consist of a bilayer of (phospho)lipids, cho- and dissociated forms of Env [31 – 33]. These fea- lesterol, and other amphipathic molecules. Various tures, which have been implicated in HIV-1 immune types of liposomes have been used to multivalently evasion [34,35], suggest that rather than exactly present viral antigens, including the FDA-approved mimicking the natural virus, a successful VLP plat- influenza (Inflexal V) and hepatitis A (Epaxal) vac- form should present more stable and more homo- cines [41 – 44]. In the context of HIV-1 Env, lip- geneous native-like trimers and at higher density osomes are an attractive nanoparticle platform as than HIV-1 itself. they allow purification of native-like Env prior to Crooks et al. [36,37] have addressed several of nanoparticle formation. these issues by first stabilizing the Env trimer by Early generations of liposomes used in HIV-1 2þ addition of a disulfide-bond (SOS) between gp120 Env research used lipids bearing Ni at the polar and gp41, then removing uncleaved and other non- head group. Purified Env trimers with a C-terminal native forms of Env from the VLP surface by sub- His-tag could then be noncovalently coupled to the jecting them to a cocktail of glycosidases and liposome. This strategy was applied to various Env proteases. The fact that, in contrast to untreated trimers and showed that the Env-liposomes were VLPs, the protease-digested VLPs were able to induce superior at B-cell activation than the parental solu- some level of heterologous Tier-2 virus neutraliza- ble trimers in vitro [8,20,45]. Furthermore, when tion, albeit inconsistently and weakly, suggests the administered to rhesus macaques, Env-liposomes importance of improving the structural homogene- were able to increase the quantities of follicular T ity of expressed trimeric Env on HIV-1 VLPs [37,38]. helper cells and proliferating B cells in germinal 304 www.co-hivandaids.com Volume 14  Number 4  July 2019 Copyright © 2019 Wolters Kluwer Health, Inc. All rights reserved. HIV-1 Env trimer nanoparticle immunogens Brouwer and Sanders centers, whereas soluble trimers were unable to do fusion of Env to ferritin yields a 24 subunit nano- so [45]. In addition, Env-liposomes induced mod- particle of around 30 nm presenting eight trimers estly higher autologous NAb responses than the [47]. Two subsequent studies by He et al. and Sliepen parental trimer. However, the noncovalent Env- et al. showed that Env trimers presented on ferritin 2þ liposome linkage through Ni and His-tag proved nanoparticles induced significantly higher binding instable in vivo as these liposomes were unable to antibody and autologous NAb responses than single & & preserve the Env trimers on their surface [6,18 ,19]. trimers in rabbits (Sliepen et al. in press) [49 ]. The To overcome the issue of Env dissociation, next- benefit of the nanoparticles was evident particularly generation Env-liposomes were developed that after the priming immunization (Sliepen et al.in contain lipids with a maleimide group allowing for press). covalent coupling of trimers using a C-terminal cys- As a higher number of Env trimers per particle teine. This strategy markedly increased the stability may further improve the immunogenicity of pro- of Env conjugation. Although Env-liposomes gener- tein nanoparticles, several one-component nano- 2þ ated using Ni -His interactions where devoid of particles have been described that present 20 Env trimers after a 4-day incubation in 20% mouse serum, trimers. Fusion of Env to E2p, a 60-subunit particle Env-liposomes generated through maleimide-cyste- from B. Stearothermophilus, yielded particles that, in ine cross-linking maintained Env on their surface contrast to ferritin nanoparticles, were able to biva- [18 ,19]. Moreover, the later types of Env-liposomes lently interact with certain bNAbs. However, the significantly increased the percentage of germinal yields of these particles was low and Env antigenic- center (GC) B cells in the draining lymph nodes of ity was not optimal [49 ]. The computationally immunized mice and induced significantly higher designed I3 – 01 nanoparticle platform has also been binding Ab titers than the former liposomes and used to present 20 Env trimers [49 ,50]. T-cell epit- soluble trimers [19]. However, despite these improve- opes to further enhance Env immunogenicity were ments, rhesus macaques immunized with a stabilized also included in these nanoparticles. Although sol- Env trimer conjugated to liposomes by maleimide- uble Env failed to induce autologous neutralization cysteine cross-linking developed significantly lower in mice, Env presented on I3 – 01 with a T-cell epi- autologous NAbs than the soluble counterpart and tope induced modest autologous NAb responses in the authors speculated that the stability of these lip- two of eight rabbits. osomes remained inadequate [6]. Env retention on Although several in vivo-assembling protein liposomes could be further enhanced by increasing nanoparticles were able to augment Env immuno- the maleimide concentration from 5 to 15%, intro- genicity, the improvements were modest and incon- ducing sphingomyelin and exchanging the lipid sistent, at best. One issue with ferritin nanoparticles DSPC to DPPC. Although an initial immunization (and presumably also E2p and I3-01 nanoparticles), study in mice showed that the increased stabilization and one that might be inherent to most in vivo- further enhanced the percentage of GC B cells, it assembling platforms, is that once the nanoparticles remains to be tested whether these liposomes are are assembled, presumably in the endoplasmic retic- able to increase NAb responses in a more relevant ulum, furin has no easy access to its cleavage site animal model [18 ]. between gp120 and gp41, resulting in incomplete precursor cleavage (Sliepen et al. in press) [47,51]. This combined with the inability to exclude nonna- In vivo-assembling protein nanoparticles tive Env trimers from the nanoparticles (Fig. 1), may The discovery and design of proteins that self-assem- lead to the exposure of highly immunodominant ble into well ordered complexes with three-fold epitopes that are irrelevant to neutralization of pri- symmetry has opened the door for the development mary, neutralization-resistant (Tier 2) viruses, and of protein-only nanoparticles presenting Env might even distract from NAb epitopes [52]. Indeed, trimers [46]. In recent years, the genetic fusion of Env-ferritin nanoparticles usually augment NAb Env to the termini of these one-component self- responses against lab-adapted and highly neutrali- assembling proteins has yielded various recombi- zation sensitive viruses (Tier 1) more than NAb nantly expressed nanoparticles that present 8 or responses against primary Tier 2 viruses, consistent 20 trimers [12,13,21,47,48,49 ]. The well ordered with the concerns described above (Sliepen et al.in geometry of the presented Env and the ease of press; Brouwer et al. in revision) [47,51]. production, which can be scaled up using current translational methods, make these nanoparticles an In vitro-assembling protein nanoparticles attractive platform. One of the first self-assembling proteins to be In vitro-assembling nanoparticle systems offer used in combination with Env is ferritin. Genetic advantages as they allow more control over the 1746-630X Copyright  2019 Wolters Kluwer Health, Inc. All rights reserved. www.co-hivandaids.com 305 Copyright © 2019 Wolters Kluwer Health, Inc. All rights reserved. Broad neutralising and non-neutralising antibodies FIGURE 1. In vitro-assembling (two-component) nanoparticles allow for greater control over Env quality than in vivo- assembling (one-component) nanoparticles. In contrast to in vivo-assembling nanoparticles such as Env-ferritin (top panel) or VLPs presenting Env, in vitro-assembling nanoparticles such as Env-liposomes or Env-I53-50 nanoparticles (bottom) allow purification of Env trimers prior to assembly ensuring that native-like trimers (green) are selected and nonnative trimers (red) are excluded from the nanoparticles. quality of the Env trimers by allowing the purifica- NAb responses were 40-fold higher in the Env-I53- tion of native-like trimers prior to assembly. The 50NP group compared to the single trimer group. recent development of protein nanoparticles that Furthermore, Env-I53-50NPs were superior over self-assemble in vitro has opened up new platform for Env-ferritin particles in augmenting autologous nanoparticle vaccines [53,54]. Bale et al. [53] used NAb responses, while suppressing responses against computational protein structure prediction to nonneutralizing epitopes, confirming the quality of design a large number of icosahedral particles which the presented Env trimers. assembled highly-efficiently in vitro. One nanopar- However, we found that the benefit of nanopar- ticle design in particular, designated I53-50 and ticle presentation effect was dependent on the loca- consisting of 20 trimeric (I53-50A) and 12 pentame- tion of the immunodominant epitope of the ric (I53-50B) subunits, was amenable for fusion to particular Env used. When Env-I53-50NPs with an && viral glycoproteins [55 ]. We fused Env trimers to immunodominant epitope proximal to the trimer the trimeric I53-50A component, and subsequent base were used, there was no beneficial effect of mixing with the pentameric I53-50B component nanoparticle presentation (Brouwer et al. in revi- produced monodisperse and well ordered nanopar- sion). This proved to be a result of poor epitope ticles presenting twenty trimers (Env-I53-50NP). accessibility and showed that epitope location and The assembly efficiency and Env quality were excel- accessibility are parameters that should be consid- lent (Brouwer et al. in revision). ered in nanoparticle vaccine design. Immunization studies in rabbits showed that Env-I53-50NPs significantly increased Env immuno- CONCLUSION genicity, including NAb responses (Brouwer et al. in revision). This was particularly evident after the Recent years have not only seen the emergence of priming immunizations, when the autologous new Env-nanoparticle platforms but also increased 306 www.co-hivandaids.com Volume 14  Number 4  July 2019 Copyright © 2019 Wolters Kluwer Health, Inc. All rights reserved. HIV-1 Env trimer nanoparticle immunogens Brouwer and Sanders 7. Torrents de la Pen ˜ a A, Julien J-P, de Taeye SW, et al. 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Acknowledgements This study revealed a novel mechanism by which glycosylated nanoparticle immunogens improve lymph node trafficking and germinal center formation, by We acknowledge Neil King for kindly sharing the Pymol using the lectin-mediated complement pathway. script that was used to the create the Env trimer and 18. Tokatlian T, Kulp DW, Mutafyan AA, et al. Enhancing humoral responses & against HIV envelope trimers via nanoparticle delivery with stabilized synthetic nanoparticle structures in Fig. 1. liposomes. Sci Rep 2018; 8:16527. Tokatlian et al. presented the design of stabilized synthetic liposomes. A systema- tic in vivo study in mice suggested liposomes are most efficacious during the Financial support and sponsorship priming stage of immunization. Work by the authors in this area is supported by the U.S. 19. Bale S, Goebrecht G, Stano A, et al. Covalent linkage of HIV-1 trimers to synthetic liposomes elicits improved B cell and antibody responses. 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Kuai R, Li D, Chen YE, et al. High-density lipoproteins: nature’s multifunctional 54. King NP, Bale JB, Sheffler W, et al. Accurate design of co-assembling nanoparticles. ACS Nano 2016; 10:3015 – 3041. multicomponent protein nanomaterials. Nature 2014; 510:103 – 108. 43. Herzog C, Hartmann K, Kunzi € V, et al. Eleven years of Inflexal V-a virosomal 55. Marcandalli J, Fiala B, Ols S, et al. Induction of potent neutralizing antibody adjuvanted influenza vaccine. Vaccine 2009; 27:4381 – 4387. && responses by a designed protein nanoparticle vaccine for respiratory syncytial 44. Bovier PA. Epaxal: a virosomal vaccine to prevent hepatitis A infection. Expert virus. Cell 2019; 176:1420 – 1431.e17. Rev Vaccines 2008; 7:1141 – 1150. Marcandalli et al. describe the structure-based design and characterization of a 45. Martinez-Murillo P, Tran K, Guenaga J, et al. Particulate array of well ordered two-component protein nanoparticle presenting RSV-F glycoproteins. This work HIV clade C Env trimers elicits neutralizing antibodies that display a unique V2 introduced a novel class of fully synthetic nanoparticles that can be assembled with Cap approach. Immunity 2017; 46:; 804-817.e7. high efficiency and controllability into well-ordered structures in vitro. 308 www.co-hivandaids.com Volume 14  Number 4  July 2019 Copyright © 2019 Wolters Kluwer Health, Inc. All rights reserved. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Current Opinion in HIV and Aids Wolters Kluwer Health

Presentation of HIV-1 envelope glycoprotein trimers on diverse nanoparticle platforms

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Downloaded from https://journals.lww.com/co-hivandaids by BhDMf5ePHKav1zEoum1tQfN4a+kJLhEZgbsIHo4XMi0hCywCX1AWnYQp/IlQrHD35y8U/jUqeEyVMweXB0LtB1dl03evjuXlpE8l2qkMB64= on 12/16/2019 REVIEW URRENT Presentation of HIV-1 envelope glycoprotein PINION trimers on diverse nanoparticle platforms a a,b Philip J.M. Brouwer and Rogier W. Sanders Purpose of review We will discuss recent advances in the development of nanoparticle vaccines presenting HIV-1 envelope trimer vaccines and the immunological mechanisms by which they act. Recent findings The multivalent presentation of Env trimers on nanoparticles is a promising strategy to increase Env immunogenicity. Recent studies have shed light on how Env nanoparticles increase lymph node trafficking and germinal center formation by using the lectin-mediated complement pathway and enhancing the interaction with naı¨ve B cells. Meanwhile, research on different nanoparticle platforms has resulted in improved designs, such as liposomes with improved stability, and the emergence of novel platforms such as protein nanoparticles that self-assemble in vitro. Immmunogenicity studies with these nanoparticles delineate the advantages and expose the limitations of the different nanoparticle platforms. Summary It is becoming increasingly clear that HIV-1 vaccine research might benefit greatly from using nanoparticles presenting Env trimers, particularly during the priming stage of immunization. Among the different nanoparticles that are being pursued, in vitro-assembling nanoparticles allow for greater control of Env quality making them a promising nanoparticle platform. Keywords HIV-1 Env trimer, liposome, nanoparticle presentation, self-assembling protein nanoparticle, virus-like particle INTRODUCTION based-design has resulted in the development of native-like Env trimers that have improved stability, With around 2 million new cases of HIV worldwide increased yield and reduced exposure of nonneutr- the need for a vaccine that can prevent infection alizing Abs [4,5,7 – 10]. Despite these improvements, remains as high as ever. To deal with the large native-like Env trimers generally induce NAbs with sequence diversity of HIV-1 it is widely accepted narrow specificity that are relatively short-lived. that a successful vaccine should induce broadly Presenting immunogens in a multivalent way on neutralizing antibodies (bNAbs) [1,2]. These bNAbs nanoparticles is a well established strategy to recognize and bind epitopes that are conserved increase their immunogenicity. Here, we review among a wide range of HIV-1 genotypes. The only the most recent advances in Env nanoparticle design target for (b)NAbs is the envelope glycoprotein and the immunological observations made using (Env), a trimer of heterodimers, consisting of three them. gp120 and three gp41 subunits that interact non- covalently. Over the years various forms of recom- binantly expressed Env have been used in attempts Department of Medical Microbiology, Amsterdam UMC, University of to generate a vaccine that could induce NAbs. An Amsterdam, Amsterdam, the Netherlands and Department of Microbi- important breakthrough was the development of ology and Immunology, Weill Medical College of Cornell University, New native-like trimers exemplified by the prototype York, New York, USA BG505 SOSIP.664 trimer [3]. These soluble and sta- Correspondence to Rogier W. Sanders, Department of Medical Micro- bilized native-like mimics of functional Env present biology, Amsterdam UMC, University of Amsterdam, 1105 AZ, Amster- on infectious virus were the first immunogens to dam, the Netherlands. Tel: +31 20 5667768; fax: +31 20 6916531; e-mail: r.w.sanders@amc.uva.nl elicit potent and consistent NAb responses against neutralization-resistant clinically relevant (Tier-2) Curr Opin HIV AIDS 2019, 14:302–308 viruses in animal models [4 – 6]. Since then, structure DOI:10.1097/COH.0000000000000549 www.co-hivandaids.com Volume 14  Number 4  July 2019 Copyright © 2019 Wolters Kluwer Health, Inc. All rights reserved. Downloaded from https://journals.lww.com/co-hivandaids by BhDMf5ePHKav1zEoum1tQfN4a+kJLhEZgbsIHo4XMi0hCywCX1AWnYQp/IlQrHD35y8U/jUqeEyVMweXB0LtB1dl03evjuXlpE8l2qkMB64= on 12/16/2019 HIV-1 Env trimer nanoparticle immunogens Brouwer and Sanders monomeric antigens [16]. Recently, Tokatlian et al. KEY POINTS provided new insights into the immunological mechanisms behind the improved trafficking to Multivalent presentation of Env trimers on nanoparticles lymph nodes of nanoparticles carrying highly gly- increases germinal center responses by improving the && cosylated antigens such as Env [17 ]. Nanoparticles trafficking to and retention in lymph nodes by a complement-dependent mechanism and by enhancing presenting Env trimers where present in lymph the activation of naı¨ve B cells. node follicles, germinal centers, and on fDCs in much higher quantities than their soluble counter- Improvements in VLP, liposome, and in vivo-assembling parts. This was shown to be largely dependent on protein nanoparticle platforms include enhanced complement, as colocalization of particles on fDCs stability and improved Env quality and valency. A new nanoparticle platform has emerged, which consists of in was significantly reduced in mice lacking comple- vitro-assembling protein nanoparticle. ment component C3 or its receptors Cr1/2. They further showed that mannose-binding lectin bound Although Env nanoparticles were generally able to significantly better to nanoparticles than soluble increase Env-binding titers, the modest and inconsistent increase of NAb titers by various nanoparticle immunogens and that the mannose-binding lectin platforms suggest that other factors (i.e. Env pathway mediated the observed fDC targeting && antigenicity, epitope accessibility) other than multivalent [17 ]. Thus, as a result of increased avidity by mul- presentation are critical for optimal exploitation of tivalent presentation of Env, nanoparticles more nanoparticle platforms. efficiently initiate the mannose-binding lectin – pathway resulting in higher deposition of comple- The ability to purify Env trimers prior to assembly in liposomes and in vitro-assembling protein nanoparticles ment. This subsequently leads to more efficient allows for control of Env quality making them promising trafficking to lymph node follicles and presentation nanoparticle platforms. on fDCs. Once in the lymph nodes, nanoparticles will bind more avidly to B-cell receptors (BCRs) on naive cognate B cells than soluble immunogens, enhanc- IMMUNOLOGICAL MECHANISMS OF ing BCR clustering, intracellular BCR-mediated sig- ACTION OF NANOPARTICLES naling, and subsequent activation of the B cell. The success of the commercially available HPV and Indeed, several studies using Env nanoparticles HBV vaccines and the promising preclinical data for and Env-specific B cells show that nanoparticle influenza, EBV and malaria nanoparticle vaccines, presentation enhances B-cell activation in vitro reinforce the paradigm that multivalent presenta- (Brouwer et al. in revision) [8,18 ,19,20,21]. The tion of an immunogen can enhance its immunoge- ability to more efficiently activate cognate B cells nicity [11 – 14]. In light of this, Env trimers have may be of particular relevance in the context of been presented on various nanoparticle platforms of HIV-1 immunogens as bNAb precursors are gener- which several have shown clear improvements in ally highly infrequent and have low affinity for Env && Env immunogenicity. Meanwhile, the immunolog- [22]. Recently, Abbott et al. [23 ] tested the effects of ical mechanisms of action of nanoparticle vaccines immunogen affinity and avidity on germinal center are being elucidated and the recent findings are formation in mice that had a range of precursor discussed here. The improved immunogenicity of frequencies of a specific germline B cell. At physio- nanoparticle immunogens is rooted in several logical affinities (0.5 mMK ) and frequencies (one in immunological processes including antigen traffick- 1  10 cells), nanoparticles were able to expand ing to lymph nodes, activation of antigen-specific B specific germline B cells whereas with the soluble cells, and uptake by and activation of antigen pre- counterpart these B cells were undetectable. Further- senting cells [15]. It is becoming increasingly clear more, even with 10-fold lower precursor frequencies that the benefit of nanoparticles is a result of the and a 16,000 lower affinity, nanoparticle immu- enhancement of multiple immunological processes nogens outcompeted the monomer with respect to involving both innate and adaptive responses. the quantity of detectable specific B cells 8 days post && To mount an antibody response, an immunogen immunization [23 ]. has to find its way into the draining lymph nodes Inducing antigen-presenting cells to migrate to where it will interact with B cells and follicular lymph nodes is a crucial process in the development dendritic cells (fDCs). In the case of direct lymphatic of a humoral response as they prime cognate follic- drainage, antigens will drain from the administra- ular T helper cells that drive B-cell affinity matura- tion site by interstitial flow towards a lymphatic tion in the germinal centers. Nanoparticles have vessel. As particulate antigens have lower diffusion been shown to increase uptake by and activation rates they will be delivered more effectively than of dendritic cells over soluble antigen, with a 1746-630X Copyright  2019 Wolters Kluwer Health, Inc. All rights reserved. www.co-hivandaids.com 303 Copyright © 2019 Wolters Kluwer Health, Inc. All rights reserved. Broad neutralising and non-neutralising antibodies marked dependency on nanoparticle size [24 – 27]. A common strategy to increase the quantity of Env The improved uptake of particles with a similar size on the VLP surface is truncating the cytoplasmic tail. to that of viruses (20 – 200 nm) has led to specula- Recently, Stano et al. [39] were able to further tions that DCs have evolved to recognize virus-sized increase Env quantity by generating a VLP produc- particles [26,28,29]. However, in the case of recep- tion platform that yielded VLPs with more than 100 tor-mediated endocytosis the higher avidity by Env trimers on the surface. How these higher nanoparticle immunogens may also play a role in valency VLPs perform in vivo remains to be the enhanced uptake [30]. To what extent and by addressed. An interesting approach to improve the which mechanism Env nanoparticles increase anti- immunogenicity of Env VLPs was introduced by gen presentation remains to be studied. Elsayed et al. [40], who generated Env VLPs that contained helper T-cell epitopes of tetanus toxoid. Pre-immunization of mice with tetanus toxoid led DIVERSE NANOPARTICLE PLATFORMS to significantly increased Env-specific IgG titers, FOR HIV-1 ENV TRIMER PRESENTATION which was shown to be caused by the intrastructural The immunological arguments presented above pro- help conferred by the helper T-cell epitope(s) [40]. vide a sound rationale for presenting Env trimers on The elegance of this approach lies in the fact that nanoparticles. Although considerable progress has humans have been vaccinated against tetanus dur- been made, several problems, including poor in vivo ing childhood and that the tetanus memory T cells stability of Env-nanoparticles, difficulties in ensuring can be harnessed to help HIV-1 Env immunogenic- and preserving native-like Env structure, and produc- ity, at least in theory. tion issues have limited the improvements in Env Despite these recent advances, producing Env immunogenicity conferred by nanoparticle presen- VLPs to yields that are sufficient for testing in larger tation so far. Below, we discuss the main nanoparticle animal models (rabbits, macaques) remains a critical platforms that are currently being explored. bottleneck. Although the yield of Env-VLPs could be increased by optimizing plasmid combinations, it was estimated that per unit time, more than 100- Virus-like particles fold less doses could be produced compared to SOSIP One of the earliest nanoparticles that have been trimers [38]. Combining the described strategies explored are virus-like particles (VLPs). These multi- that enhance trimeric integrity and quantity of protein, enveloped particles are produced by presented Env and finding ways to increase VLP cotransfecting the gag and env gene, resulting in expression will probably be crucial to solve the the formation of replication-incompetent particles multiple limitations Env VLPs have. that mimic the shape and conformation of the HIV- 1 virus. However, HIV-1 particles generally only Liposomes present on average 7 – 14 Env molecules per particle of which a substantial fraction is nonfunctional Liposomes are unilamellar or multilamellar vesicles, with a nonnative structure, including uncleaved which consist of a bilayer of (phospho)lipids, cho- and dissociated forms of Env [31 – 33]. These fea- lesterol, and other amphipathic molecules. Various tures, which have been implicated in HIV-1 immune types of liposomes have been used to multivalently evasion [34,35], suggest that rather than exactly present viral antigens, including the FDA-approved mimicking the natural virus, a successful VLP plat- influenza (Inflexal V) and hepatitis A (Epaxal) vac- form should present more stable and more homo- cines [41 – 44]. In the context of HIV-1 Env, lip- geneous native-like trimers and at higher density osomes are an attractive nanoparticle platform as than HIV-1 itself. they allow purification of native-like Env prior to Crooks et al. [36,37] have addressed several of nanoparticle formation. these issues by first stabilizing the Env trimer by Early generations of liposomes used in HIV-1 2þ addition of a disulfide-bond (SOS) between gp120 Env research used lipids bearing Ni at the polar and gp41, then removing uncleaved and other non- head group. Purified Env trimers with a C-terminal native forms of Env from the VLP surface by sub- His-tag could then be noncovalently coupled to the jecting them to a cocktail of glycosidases and liposome. This strategy was applied to various Env proteases. The fact that, in contrast to untreated trimers and showed that the Env-liposomes were VLPs, the protease-digested VLPs were able to induce superior at B-cell activation than the parental solu- some level of heterologous Tier-2 virus neutraliza- ble trimers in vitro [8,20,45]. Furthermore, when tion, albeit inconsistently and weakly, suggests the administered to rhesus macaques, Env-liposomes importance of improving the structural homogene- were able to increase the quantities of follicular T ity of expressed trimeric Env on HIV-1 VLPs [37,38]. helper cells and proliferating B cells in germinal 304 www.co-hivandaids.com Volume 14  Number 4  July 2019 Copyright © 2019 Wolters Kluwer Health, Inc. All rights reserved. HIV-1 Env trimer nanoparticle immunogens Brouwer and Sanders centers, whereas soluble trimers were unable to do fusion of Env to ferritin yields a 24 subunit nano- so [45]. In addition, Env-liposomes induced mod- particle of around 30 nm presenting eight trimers estly higher autologous NAb responses than the [47]. Two subsequent studies by He et al. and Sliepen parental trimer. However, the noncovalent Env- et al. showed that Env trimers presented on ferritin 2þ liposome linkage through Ni and His-tag proved nanoparticles induced significantly higher binding instable in vivo as these liposomes were unable to antibody and autologous NAb responses than single & & preserve the Env trimers on their surface [6,18 ,19]. trimers in rabbits (Sliepen et al. in press) [49 ]. The To overcome the issue of Env dissociation, next- benefit of the nanoparticles was evident particularly generation Env-liposomes were developed that after the priming immunization (Sliepen et al.in contain lipids with a maleimide group allowing for press). covalent coupling of trimers using a C-terminal cys- As a higher number of Env trimers per particle teine. This strategy markedly increased the stability may further improve the immunogenicity of pro- of Env conjugation. Although Env-liposomes gener- tein nanoparticles, several one-component nano- 2þ ated using Ni -His interactions where devoid of particles have been described that present 20 Env trimers after a 4-day incubation in 20% mouse serum, trimers. Fusion of Env to E2p, a 60-subunit particle Env-liposomes generated through maleimide-cyste- from B. Stearothermophilus, yielded particles that, in ine cross-linking maintained Env on their surface contrast to ferritin nanoparticles, were able to biva- [18 ,19]. Moreover, the later types of Env-liposomes lently interact with certain bNAbs. However, the significantly increased the percentage of germinal yields of these particles was low and Env antigenic- center (GC) B cells in the draining lymph nodes of ity was not optimal [49 ]. The computationally immunized mice and induced significantly higher designed I3 – 01 nanoparticle platform has also been binding Ab titers than the former liposomes and used to present 20 Env trimers [49 ,50]. T-cell epit- soluble trimers [19]. However, despite these improve- opes to further enhance Env immunogenicity were ments, rhesus macaques immunized with a stabilized also included in these nanoparticles. Although sol- Env trimer conjugated to liposomes by maleimide- uble Env failed to induce autologous neutralization cysteine cross-linking developed significantly lower in mice, Env presented on I3 – 01 with a T-cell epi- autologous NAbs than the soluble counterpart and tope induced modest autologous NAb responses in the authors speculated that the stability of these lip- two of eight rabbits. osomes remained inadequate [6]. Env retention on Although several in vivo-assembling protein liposomes could be further enhanced by increasing nanoparticles were able to augment Env immuno- the maleimide concentration from 5 to 15%, intro- genicity, the improvements were modest and incon- ducing sphingomyelin and exchanging the lipid sistent, at best. One issue with ferritin nanoparticles DSPC to DPPC. Although an initial immunization (and presumably also E2p and I3-01 nanoparticles), study in mice showed that the increased stabilization and one that might be inherent to most in vivo- further enhanced the percentage of GC B cells, it assembling platforms, is that once the nanoparticles remains to be tested whether these liposomes are are assembled, presumably in the endoplasmic retic- able to increase NAb responses in a more relevant ulum, furin has no easy access to its cleavage site animal model [18 ]. between gp120 and gp41, resulting in incomplete precursor cleavage (Sliepen et al. in press) [47,51]. This combined with the inability to exclude nonna- In vivo-assembling protein nanoparticles tive Env trimers from the nanoparticles (Fig. 1), may The discovery and design of proteins that self-assem- lead to the exposure of highly immunodominant ble into well ordered complexes with three-fold epitopes that are irrelevant to neutralization of pri- symmetry has opened the door for the development mary, neutralization-resistant (Tier 2) viruses, and of protein-only nanoparticles presenting Env might even distract from NAb epitopes [52]. Indeed, trimers [46]. In recent years, the genetic fusion of Env-ferritin nanoparticles usually augment NAb Env to the termini of these one-component self- responses against lab-adapted and highly neutrali- assembling proteins has yielded various recombi- zation sensitive viruses (Tier 1) more than NAb nantly expressed nanoparticles that present 8 or responses against primary Tier 2 viruses, consistent 20 trimers [12,13,21,47,48,49 ]. The well ordered with the concerns described above (Sliepen et al.in geometry of the presented Env and the ease of press; Brouwer et al. in revision) [47,51]. production, which can be scaled up using current translational methods, make these nanoparticles an In vitro-assembling protein nanoparticles attractive platform. One of the first self-assembling proteins to be In vitro-assembling nanoparticle systems offer used in combination with Env is ferritin. Genetic advantages as they allow more control over the 1746-630X Copyright  2019 Wolters Kluwer Health, Inc. All rights reserved. www.co-hivandaids.com 305 Copyright © 2019 Wolters Kluwer Health, Inc. All rights reserved. Broad neutralising and non-neutralising antibodies FIGURE 1. In vitro-assembling (two-component) nanoparticles allow for greater control over Env quality than in vivo- assembling (one-component) nanoparticles. In contrast to in vivo-assembling nanoparticles such as Env-ferritin (top panel) or VLPs presenting Env, in vitro-assembling nanoparticles such as Env-liposomes or Env-I53-50 nanoparticles (bottom) allow purification of Env trimers prior to assembly ensuring that native-like trimers (green) are selected and nonnative trimers (red) are excluded from the nanoparticles. quality of the Env trimers by allowing the purifica- NAb responses were 40-fold higher in the Env-I53- tion of native-like trimers prior to assembly. The 50NP group compared to the single trimer group. recent development of protein nanoparticles that Furthermore, Env-I53-50NPs were superior over self-assemble in vitro has opened up new platform for Env-ferritin particles in augmenting autologous nanoparticle vaccines [53,54]. Bale et al. [53] used NAb responses, while suppressing responses against computational protein structure prediction to nonneutralizing epitopes, confirming the quality of design a large number of icosahedral particles which the presented Env trimers. assembled highly-efficiently in vitro. One nanopar- However, we found that the benefit of nanopar- ticle design in particular, designated I53-50 and ticle presentation effect was dependent on the loca- consisting of 20 trimeric (I53-50A) and 12 pentame- tion of the immunodominant epitope of the ric (I53-50B) subunits, was amenable for fusion to particular Env used. When Env-I53-50NPs with an && viral glycoproteins [55 ]. We fused Env trimers to immunodominant epitope proximal to the trimer the trimeric I53-50A component, and subsequent base were used, there was no beneficial effect of mixing with the pentameric I53-50B component nanoparticle presentation (Brouwer et al. in revi- produced monodisperse and well ordered nanopar- sion). This proved to be a result of poor epitope ticles presenting twenty trimers (Env-I53-50NP). accessibility and showed that epitope location and The assembly efficiency and Env quality were excel- accessibility are parameters that should be consid- lent (Brouwer et al. in revision). ered in nanoparticle vaccine design. Immunization studies in rabbits showed that Env-I53-50NPs significantly increased Env immuno- CONCLUSION genicity, including NAb responses (Brouwer et al. in revision). This was particularly evident after the Recent years have not only seen the emergence of priming immunizations, when the autologous new Env-nanoparticle platforms but also increased 306 www.co-hivandaids.com Volume 14  Number 4  July 2019 Copyright © 2019 Wolters Kluwer Health, Inc. All rights reserved. HIV-1 Env trimer nanoparticle immunogens Brouwer and Sanders 7. Torrents de la Pen ˜ a A, Julien J-P, de Taeye SW, et al. 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Acknowledgements This study revealed a novel mechanism by which glycosylated nanoparticle immunogens improve lymph node trafficking and germinal center formation, by We acknowledge Neil King for kindly sharing the Pymol using the lectin-mediated complement pathway. script that was used to the create the Env trimer and 18. Tokatlian T, Kulp DW, Mutafyan AA, et al. Enhancing humoral responses & against HIV envelope trimers via nanoparticle delivery with stabilized synthetic nanoparticle structures in Fig. 1. liposomes. Sci Rep 2018; 8:16527. Tokatlian et al. presented the design of stabilized synthetic liposomes. A systema- tic in vivo study in mice suggested liposomes are most efficacious during the Financial support and sponsorship priming stage of immunization. Work by the authors in this area is supported by the U.S. 19. Bale S, Goebrecht G, Stano A, et al. Covalent linkage of HIV-1 trimers to synthetic liposomes elicits improved B cell and antibody responses. 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This work HIV clade C Env trimers elicits neutralizing antibodies that display a unique V2 introduced a novel class of fully synthetic nanoparticles that can be assembled with Cap approach. Immunity 2017; 46:; 804-817.e7. high efficiency and controllability into well-ordered structures in vitro. 308 www.co-hivandaids.com Volume 14  Number 4  July 2019 Copyright © 2019 Wolters Kluwer Health, Inc. All rights reserved.

Journal

Current Opinion in HIV and Aids – Wolters Kluwer Health

Published: Jul 1, 2019

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