Research Collection
Doctoral Thesis
Design and Evaluation of a Self-Assembling Nanoparticle Vaccine against Human Cytomegalovirus
Author(s):
Perotti, Michela Publication Date:
2020
Permanent Link:
https://doi.org/10.3929/ethz-b-000449911
Rights / License:
In Copyright - Non-Commercial Use Permitted
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DISS. ETH NO 27175
DESIGN AND EVALUATION OF A SELF- ASSEMBLING NANOPARTICLE VACCINE
AGAINST HUMAN CYTOMEGALOVIRUS
A thesis submitted to obtain the degree of DOCTOR OF SCIENCES of ETH ZURICH
(Dr. sc. ETH Zurich)
presented by
MICHELA PEROTTI
MSc. Veterinary Biotechnology Sciences University of Milan, Italy
Born on 27.01.1992 in Chiavenna, Sondrio, Italy
accepted on the recommendation of
Prof. Dr. Federica Sallusto (examiner)
Prof. Dr. Laurent Perez (co-examiner)
Prof. Dr. Annette Oxenius (co-examiner)
Prof. Dr. Martin Bachman (co-examiner)
Alla mia famiglia, tutta quanta
Table of Contents
Summary 1
Summary (English) 1
Riassunto (Italiano) 3
Table of Abbreviations 5
1 Introduction 8
1.1 Human Cytomegalovirus (HCMV) 8
1.1.1 HCMV Pathogenesis 8
1.1.2 HCMV Genome 9
1.1.3 HCMV Entry and Replication 9
1.1.4 HCMV Glycoproteins 11
1.1.4.1 The Glycoprotein B (gB) 12
1.1.4.2 The Trimeric gHgLgO Complex 14
1.1.4.3 The Pentameric gHgLpUL128/130/131A Complex 15
1.2 Immune Control of HCMV Infection 17
1.2.1 Innate Immune Response 19
1.2.2 Cellular Immune Response 19
1.2.3 Humoral Immune Response 21
1.2.4 Viral Immune Evasion 22
1.3 Vaccines against HCMV 24
1.3.1 Live-Attenuated Vaccines 25
1.3.2 Recombinant gB Subunit Vaccines 25
1.3.3 Viral Vectored Vaccines 26
1.3.4 DNA/mRNA-Based Vaccines 27
1.3.5 Virus-Like Particle Vaccines 28
1.4 Self-Assembling Nanoparticle for Antigen Display 29
1.4.1 Ferritin-Based Nanoparticle 30
1.4.2 De novo Design-Based Nanoparticle 32
2 Aim of the Thesis 35
3 Results 36
4 Discussion and Outlook 68
5 References 73
6 Curriculum Vitae 90
7 List of Publications 92
Acknowledgments 177
Summary
Summary (English)
The Human Cytomegalovirus (HCMV) is a ubiquitously distributed member of the Herpesviridae family that establishes lifelong infection in humans. Seroprevalence in the human adult population is superior to 70% worldwide. HCMV is responsible for high morbidity and mortality in immunocompromised patients, and is the leading viral cause of congenital birth defects, affecting 0.7% of newborns with permanent sequelae such as mental retardation and hearing loss. The development of a HCMV vaccine has been deemed a national priority by the US National Academy of Medicine since 1999. Several clinical trials targeting different HCMV antigens, such as the glycoprotein (g)B, the pentameric complex or the phosphoprotein 65 (pp65), have been conducted. However, none of those conferred full sterilizing immunity and up to date there is no vaccine or monoclonal antibody (mAb) approved for clinical use.
The employment of neutralizing antibodies to identify protective antigens, in association with structural vaccinology to stabilize an antigen of interest and scaffold presentation, to enhance immunogenicity, are required to target challenging pathogens such as HCMV. With a general methodology for rational vaccine design, combining identification of prevailing antigenic domains (ADs) with orthogonal technologies, we aimed at generating a HCMV candidate vaccine with an increased immunogenicity.
Applying our approach to the immunogenic gB fusogen, we have not only characterized the immunogenicity of its individual ADs, but also demonstrated that gB domain I (AD5) is the main target of neutralizing antibodies. Through sophisticated antigen trimerization and stabilization techniques, to ensure proper antigen conformation, we were able to focus the antibody response to the key protective domain of the HCMV fusion glycoprotein. To cope with the ambitious purpose of further increase the immunogenicity of our candidate vaccine, we employed the multivalent antigen presentation properties of nanoparticle scaffolds.
Indeed, presentation of multiple copies of an antigen in a repetitive array is known to drive a more robust humoral immune response than its soluble counterpart. Fusion of our stabilized antigen with different carriers allowed the generation of two HCMV candidate vaccines displaying multiple copies of the trimeric gB-AD5 antigen. We showed that
mice, compared to the most studied recombinant gB vaccines. Collectively, these results illustrate with a medically relevant example, a general approach combining antigen discovery, protein engineering and scaffold presentation for modern development of subunit vaccines against complex pathogens.
Riassunto (Italiano)
Il citomegalovirus umano (HCMV) è un virus appartenente alla famiglia degli Herpesviridae, estremamente diffuso a livello globale. Una volta contratto, l’HCMV rimane latente all’interno dell’organismo per tutta la vita, ma può riattivarsi in caso di indebolimento del sistema immunitario. Si stima che più del 70% della popolazione mondiale adulta vada incontro ad un’infezione da HCMV. Sebbene di norma l’infezione si evolva senza sintomi, negli individui immunocompromessi il virus può causare un’elevata morbilità e mortalità. Inoltre, l’aspetto più importante legato all’HCMV, è rappresentato dall’essere la principale causa virale di infezioni congenite. Un’infezione contratta durante la gravidanza e trasmessa al feto può, nello 0.7% dei casi, arrecare danni permanenti quali, per esempio, ritardo mentale e perdita dell’udito. Dato il grande impatto sociale dell’infezione da HCMV, nel 1999 un’analisi condotta dall’US Institute of Medicine posizionò lo sviluppo di un vaccino in cima alla lista di priorità. Da allora, numerosi vaccini, basati su antigeni dell’HCMV quali la glicoproteina B (gB), il complesso pentamerico e la fosfoproteina 65 (pp65), sono stati sviluppati e testati in studi clinici. Tuttavia, nessuno di questi è stato in grado di conferire una protezione completa, pertanto, ad oggi non esiste alcun vaccino o anticorpo monoclonale approvato per l’uso clinico.
Nel caso di patogeni critici, come l’HCMV, i classici approcci per lo sviluppo di vaccini non sono sempre sufficienti. In questi casi, l’utilizzo di molteplici strategie come l’identificazione di antigeni protettivi tramite anticorpi in grado di neutralizzare l’infezione virale, le analisi strutturali per stabilizzare gli antigeni d’interesse e la loro corretta presentazione per migliorarne l'immunogenicità si rivelano necessari. Il nostro approccio, mirato a generare un nuovo e migliorato vaccino contro l’HCMV, si è basato su una progettazione razionale che combinava l’identificazione degli antigeni virali dominanti con l’ingegneria proteica. Applicando la nostra strategia alla ben nota ed immunogenica proteina di fusione gB, abbiamo non solo caratterizzato il ruolo nell’induzione di una risposta anticorpale da parte dei suoi singoli domini antigenici (AD), ma anche dimostrato che l’AD denominato 5 (AD5) è il target principale degli anticorpi neutralizzanti generati. Attraverso una sofisticata tecnica di stabilizzazione e trimerizzazione dell’antigene, volta a garantirne la corretta conformazione, siamo stati in grado di focalizzare ed intensificare la risposta anticorpale al dominio immunogenico chiave della glicoproteina di fusione dell’HCMV. Per
vaccino, abbiamo sfruttato le proprietà immunogeniche delle Nanoparticle usate come vettori. È infatti risaputo che, la presentazione ordinata di molteplici copie dello stesso antigene sulla superficie esterna del nucleo di Nanoparticle, determina una risposta immunitaria potenziata rispetto al medesimo antigene somministrato singolarmente. La fusione della versione stabilizzata di AD5 con diversi vettori ha permesso di selezionare due Nanoparticle, entrambe caratterizzate dalla presenza di più copie dell’antigene sulla loro superficie. Tramite l’immunizzazione di topi, abbiamo dimostrato che le nostre due AD5- Nanoparticle sono in grado di suscitare un titolo anticorpale capace di bloccare l’infezione di HCMV, 100 volte superiore rispetto a quello indotto dal vaccino basato unicamente sulla proteina di fusione gB. L’insieme di questi risultati dimostra, con un esempio clinicamente rilevante, come la combinazione di molteplici tecniche sia necessaria per lo sviluppo di nuovi vaccini in grado di far fronte a patogeni insidiosi, per i quali, ancora non esistono terapie di prevenzione e controllo efficaci.
Table of Abbreviations
AD Antigenic Domain
ADCC Antibody-Dependent Cell mediated Cytotoxicity ADCP Antibody-Dependent Cellular Phagocytosis
BCR B Cell Receptor
cryoET cryo-Electron Tomography
CTL Cytotoxic T Lymphocyte
Cyto Cytoplasmic Region
E Early genes
EBV Epstein-Barr Virus
EGFR Epidermal Growth Factor Receptor
EM Electron Microscopy
ER Endoplasmic Reticulum
Fab Antigen-Binding Fragment
FCAR Immunoglobulin Alpha Fc Receptor
gB Glycoprotein B
gH Glycoprotein H
gL Glycoprotein L
gM Glycoprotein M
gN Glycoprotein N
gO Glycoprotein O
gp41 Glycoprotein 41
HBsAg Hepatitis B virus surface Antigen
HBV Hepatitis B Virus
HCMV Human Cytomegalovirus
HHV-6A Human Herpes Virus 6 A HHV-6B Human Herpes Virus 6 B
HHV-7 Human Herpes Virus 7
HHV-8 Human Herpes Virus 8
HIV Human Immunodeficiency Virus
HLTF Helicase Like Transcription Factor
HSCT Hematopoietic Stem Cells Transplant Patients HSV-1 Herpes Simplex Virus 1
HSV-2 Herpes Simplex Virus 2
IE Immediate Early genes
IE1 Immediate Early 1 protein
IE2 Immediate Early 2 protein
IFNγ Interferon-γ
IRF3 Interferon Regulatory Factor 3
L Late genes
LAcmvIL-10 HCMV viral IL-10 homologue
LILRB3 Leukocyte Ig-Like Receptor subfamily B member 3 LUNA Latency Unique Natural Antigen
mAbs monoclonal Antibodies
MCMV Murine Cytomegalovirus
MPR Membrane Proximal Region
MS Mass Spectrometry
MVA Modified Vaccinia Virus Ankara nAbs neutralizing Antibodies
NHP Nonhuman Primate
NK Natural Killer cells
NRG2 Neuregulin-2
Nrp2 Neuropilin 2
ORF Open Reading Frame
PDGFRα Platelet-Derived Growth Factor Receptor Alpha
pp65 65 kDa phosphoprotein
RBD Receptor Binding Domain
RCT Random Conical Tilt
RSV Respiratory Syncytial Virus
SOT Solid Organ Transplant
SP Signal Peptide
STING Stimulator of Interferon Genes
TGFβRIII Transforming Growth Factor Beta Receptor type 3
THBD Thrombomodulin
TLR2 Toll-Like Receptor 2
TM Transmembrane region
TNFα Tumor Necrosis Factor-α
VBI Variation Biotechnologies vaccine Incorporated VEE Venezuelan Equine Encephalitis virus
VLP Virus-Like Particle
VRP Venezuelan equine encephalitis Replicon Particle VSV Vesicular Stomatitis Virus
VZV Varicella-Zoster Virus
1 Introduction
1.1 Human Cytomegalovirus (HCMV) 1.1.1 HCMV Pathogenesis
The human Cytomegalovirus (HCMV) is a ubiquitously distributed member of the Betaherpesvirinae subfamily that establishes lifelong infection in humans. HCMV sero- prevalence rate ranges from 45% to 90% of the human adult population worldwide (Kenneson and Cannon, 2007; Plotkin et al., 2020), depending on the geographic area and socioeconomic status (Cannon et al., 2010). Virus spread occurs through body fluids including saliva, urine, semen and breast milk, vertical transmission (trans-placental), and through blood transfusion and organ transplantation (Bowden, 1991; Lang and Kummer, 1975; Reynolds et al., 1973). Primary HCMV infection in immunocompetent individuals is usually asymptomatic. However, infection or viral reactivation is an important cause of morbidity and mortality among immunocompromised individuals including AIDS patients and solid organ (SOT) or hematopoietic stem cells (HSCT) transplant recipients (Gerna et al., 1998; Lilleri and Gerna, 2016). Furthermore, HCMV is the most common viral cause of congenital birth defects affecting 0.7% of newborns with permanent sequelae such as sensorineural hearing loss, growth restriction and cognitive disabilities (Boppana and Britt, 1995). HCMV establishes a life-long infection in the host sequentially subdivided into an acute, persistence and latency phase. The acute phase lasts several days and is characterized by vigorous lytic virus replication in all tissues. When the virus replication turns from visceral organs to mucosal tissues, allowing for prolonged horizontal spread via mucosal secretions, the virus enters the persistence phase. Finally, HCMV enters latency, characterized by the retention of the viral genome in cells (mainly in myeloid cells and secretory glands) without the expression of lytic genes. The mechanisms behind the ability of the virus to reactivate the lytic replication cycle in immunocompetent hosts are not entirely understood but evidence suggests that physical trauma, inflammatory stimuli, and temporary immune suppression are involved (Fishman, 2013; Landais and Nelson, 2013).
1.1.2 HCMV Genome
HCMV belongs to the Herpesviridae family, divided into the Alpha- (herpes simplex virus 1 and 2 (HSV-1 and 2), and varicella-zoster virus (VZV)), Beta- (CMV, human herpes virus 6 A/B and 7 (HHV-6A and B and HHV-7)), and Gamma-herpesvirinae (Epstein-Barr virus (EBV) and human herpes virus 8 (HHV-8)) subfamilies. HCMV is an enveloped virus, with an icosahedral nucleocapsid that encloses a double-stranded DNA genome. The virus has one of the largest genomes, spanning approximately 200 Kbp, encoding at minimum 170 open reading frames (ORFs) and expressing more than 80 viral proteins, including glycoproteins, phosphoproteins and other transcription/replication proteins.
1.1.3 HCMV Entry and Replication
When HCMV enters the human body, it rapidly and effectively penetrates virtually all cell types, ranging from epithelial, endothelial, fibroblasts, myeloid hematopoietic precursor, and monocytes/macrophages to smooth muscle cells, stromal cells, neurons, astrocytes, hepatocytes and glial cells (Nguyen and Kamil, 2018). This penetration is accomplished by a pH-independent fusion in the case of most cells, such as fibroblast, or, as the case of epithelial cells, it occurs through endocytosis and fusion at low-pH (Compton et al., 1992).
Entry into host cells requires viral binding to specific receptors followed by the coordinated action of multiple viral entry glycoproteins (described in the next chapter) to trigger membrane fusion (Figure 1). After the virus penetrates the cell, it replicates and matures in the nucleus. All members of the Betaherpesvirinae have a relatively slow replication cycle, resulting in the characteristic multinucleated giant host cells, hence the name cytomegalia.
HCMV productive infection initiates sequential viral gene expression in three stages, starting with regulatory immediate early (IE) genes, followed by early (E) genes and concluded by late (L) genes. These three gene groups will also name the different types of HCMV proteins. IE genes transcription occurs within the first four hours after viral infection/reactivation. Accordingly, IE proteins are key regulators allowing the take-over of the cellular machinery. IE1 and IE2 are the most abundantly expressed proteins in the initial phase; activating both viral and cellular genes in the HCMV infected cells (Heider et al., 2002; Mocarski et al., 1996). Transcription of the E genes is dependent on the expression of functional IE gene products. E proteins are involved in viral replication and include DNA
transcription occurs 24 h after infection/reactivation and includes production of structural and maturation proteins. The viral products are then assembled and packaged in the nucleus whereas the final maturation and budding of the virus takes place in a Golgi-derived vacuole (Severi et al., 1988) from which the virus is released. Peculiarly, at least in vitro, only certain cells, such as fibroblasts and fully differentiated macrophages, allow a complete permissive replication cycle that results in the production of infectious viruses (Ibanez et al., 1991).
Other cells, such as monocytes, are non-permissive, meaning that the HCMV infection is restricted to the early events of gene expression and does not result in the production of complete infectious viruses (Sinclair et al., 1992). Reactivation of latent HCMV is also restricted to certain cell types and has thus far been shown to occur only in fully differentiated monocytes/macrophages (Soderberg-Naucler et al., 1997).
Figure 1. Model of HCMV viral entry.
Model for HCMV entry in human cells using the trimer and pentamer complexes. HCMV trimer interacts with PDGFRα via its gO subunit and induces the activation of gB homotrimer at the plasma membrane (Kabanova et al., 2016). The pentamer binds Nrp2, promoting endocytosis of the viral particles (Martinez-Martin et al., 2018). The release of the viral particles from the endosomal compartment could be mediated by acidification of the vesicles. Modified from Martinez-Martin et al., 2018.
1.1.4 HCMV Glycoproteins
HCMV expresses over 80 viral proteins and encodes at least 20 membrane glycoproteins on the virion envelope, which are involved in immune evasion, cell attachment and viral entry (Krishna et al., 2018). HCMV entry receptor binding and membrane fusion functions are performed by distinct viral glycoproteins. These glycoproteins are either conserved among all herpesvirus, or specific to the Betaherpesvirinae subfamily (Griffiths, 2009). The heterodimer formed by gHgL (UL75 and UL115) and the viral fusion homotrimeric protein gB (UL55) represent a core set of entry glycoproteins that is required for all herpesviruses.
In contrast, HCMV receptor binding is mediated by two distinct glycoprotein complexes: a trimeric complex composed by gO (UL74) and gHgL or a pentameric complex composed by pUL128, pUL130, pUL131A, and gHgL (Figure 2).
Figure 2. Structural representation of HCMV glycoproteins.
Structural representation of HCMV glycoproteins required for viral binding and fusion with host cellular membrane. Shown, from left to right, is the trimer, composed of the gHgL heterodimer (in green and blue respectively) disulfide-linked to gO. The pentamer is composed by the gHgL heterodimer and three additional subunits pUL128, pUL130, and pUL131A (in purple, yellow and pink respectively). The gB homotrimer is shown in its postfusion conformation. From Perotti and
1.1.4.1 The Glycoprotein B (gB)
The HCMV gB is a trimeric single-pass transmembrane protein with four extracellular domains, known to be highly conserved among the Herpesviridae family. The gB envelope glycoprotein is the proximal mediator of membrane fusion events during viral entry. HCMV gB, which is encoded by UL55, is synthesized as a 160 kDa precursor that undergoes furin cleavage in the Golgi, resulting in 116 kDa and 55 kDa fragments that remain disulfide- linked to each other (Britt and Auger, 1986). Together with the viral fusion proteins of HSV- 1, EBV and of the unrelated viruses, vesicular stomatitis virus (VSV) G and baculovirus gp64, HCMV gB, own to the class III viral fusogens (Harrison, 2008). As the others class III viral fusion proteins, gB is thought to dramatically rearrange during membrane fusion, from a metastable pre-fusion state to a stable post-fusion state (Halldorsson et al., 2018).
This conformational change brings opposing membranes in contact using energy from the protein refolding process (Harrison, 2008). The post-fusion structure of HCMV gB was published in 2015 (Burke and Heldwein, 2015; Chandramouli et al., 2015), while a recent cryo-electron tomography (cryoET) study allowed the visualization, at up to 21Å resolution, of both the pre- and post-fusion state of gB on the envelope of native HCMV virions (Si et al., 2018). The pre-fusion gB homotrimer was described flatter (extending 130 Å out from the membrane) compared to post-fusion homotrimers, which appeared taller and more columnar (extending 161 Å from the membrane). As all herpesviruses, HCMV core fusion machinery is composed of gB together with gHgL dimer, notion supported by the strictly interaction between gB and gH (Si et al., 2018; Vanarsdall et al., 2016). gB was initially reported to bind cell-surface proteins such as the epidermal growth factor receptor (EGFR) (Wang et al., 2003), and the platelet-derived growth factor receptor alpha (PDGFRα) (Soroceanu et al., 2008). However, it is more likely that gB functions as a viral fusogen that is triggered when viral and host membranes are in close proximity (Wille et al., 2013). The gB homotrimer contains five antigenic domains (ADs), defined by antibodies binding to the latter (Chandramouli et al., 2015) (Figure 3). AD1 relates to the structural domain IV and is an immune-dominant region of gB (Schoppel et al., 1997). AD2 corresponds to the N- terminal fragment and was originally defined between residues 27 and 84 (Meyer et al., 1990; Meyer et al., 1992). AD3 is the cytosolic domain of gB and, unsurprisingly, it is known to generate exclusively non-neutralizing antibodies (Kniess et al., 1991; Silvestri et
al., 2011; Spindler et al., 2014; Spindler et al., 2013). Finally, AD5 corresponds to the structural domain I (Potzsch et al., 2011). Peculiarly, mutations in each of the five gB domains prevent fusion activity, suggesting that multiple regions of gB participate in the conformational changes required for fusion and/or interactions with the proteins that trigger gB (Lin and Spear, 2007; Reimer et al., 2009). The formation of a fusion pore likely requires multiple gB trimers. Indeed, lateral interactions among gB trimers have been observed by electron microscopy (EM) and may contribute to the expansion of the fusion pore (Maurer et al., 2013).
Figure 3. Glycoprotein B (gB) structure.
(A) Schematic representation of the linear full-length gB polypeptide. Domains I-V highlighted in different colors, and the numbers of the starting residues are given. SP, signal peptide; MPR, membrane proximal region; TM, transmembrane region; Cyto, cytoplasmic domain. 698 indicates the last C-terminal residue in the gB ectodomain construct. (B) Crystal structure of one protomer of the post-fusion HCMV gB ectodomain (PDB: 5CXF) (Burke et al., 2015) that is shown as a ribbon.
The domains of gB are colored as in (A). AD2 is not shown in this representation, since its crystal structure is still to be solved. (C) The crystal structure of monomeric post-fusion gB ectodomain (left, PDB: 5CXF) is shown as ribbon next to the predicted pre-fusion gB structure (right) with domains arranged according to those in the prefusion VSV G (Roche et al., 2007). Modified from Si et al., 2018.
gB full-length A
B C
1.1.4.2 The Trimeric gHgLgO Complex
The Trimeric gHgLgO, or trimer, is a heterotrimeric complex, in which the heterodimer composed by gHgL is disulfide-liked to the betaherpesviruses-specific gO subunit, a heavily N-glycosylated polypeptide encoded by UL74 (Ciferri et al., 2015a; Huber and Compton, 1998). The trimer is one of the two viral ligands responsible for the broad cellular tropism of HCMV. Viral entry into all cells, including fibroblast, epithelial, and endothelial cells, requires gHgL in a trimeric complex with gO (Kabanova et al., 2016; Zhou et al., 2015).
Instead, the pentamer complex, composed of gHgL heterodimer and pUL128, pUL130, pUL131A subunits, is further required for efficient targeting of HCMV to epithelial and endothelial cells (Ryckman et al., 2006; Wang and Shenk, 2005). Three independent studies identified PDGFRα as the cellular receptor for the trimer (Figure 4) (Kabanova et al., 2016;
Stegmann et al., 2017; Wu et al., 2017). However, other additional receptors, including the transforming growth factor beta-receptor type 3 (TGFβRIII) and neuregulin-2 (NRG2), have been shown to bind the trimer (Martinez-Martin et al., 2018). gO correct expression and incorporation in the trimer, is of utmost importance for the functionality of the complex.
Indeed, gO null mutant fails to incorporate gHgL into the virion envelope and the resulting virus is unable to enter fibroblasts (Wille et al., 2010). Surprisingly, the trimeric complex possesses a low mean percentage of identity among the HCMV strains. While gH and gL glycoproteins are well conserved, the low degree of conservation is dictated by gO, that owns a mean identity of only 81% (Foglierini et al., 2019). Interestingly, most of the sequence divergence is located in the N-terminus part of the protein, which is supposed to interact with PDGFRα (Stegmann et al., 2017; Stegmann et al., 2019). Although the gHgLgO trimer has not been determined using crystallography, the trimer has been visualized using EM, showing that gO docks at the tip of gHgL (Figure 4) (Ciferri et al., 2015a; Kabanova et al., 2016).
Figure 4. The HCMV gHgLgO trimeric complex.
(A) Negative-stain electron microscopy and reference free 2D class averages of gHgLgO in complex with PDGFRα. The image of gHgLgO trimer-PDGFRα complex revealed that only the gO subunit interacts with PDGFRα. From Kabanova et al., 2016. (B) Random Conical Tilt (RCT) reconstruction of the gHgLgO in complex with 3G16 and 13H11 antigen-binding fragments (Fabs). The model of Ciferri et al. was based on HSV-2 gHgL dimer crystal structure, fitted into the density map. From Ciferri et al., 2015.
1.1.4.3 The Pentameric gHgLpUL128/130/131A Complex
The second gHgL complex was discovered only in 2005, after the repair of a frame-shift mutation in UL131A of the fibroblast-adapted laboratory HCMV strain AD169, restoring its infectivity for epithelial and endothelial cells (Wang and Shenk, 2005; Wang et al., 2005).
The pentameric complex is composed of the gHgL heterodimer bound to three additional glycoproteins encoded by pUL128, pUL130 and pUL131A (Ciferri et al., 2015b; Ryckman et al., 2008). Structural studies revealed that the pentamer is an elongated molecule with pULs binding at the N-terminal end of gHgL. Remarkably, comparison with the trimer structure revealed that pULs and gO bind to the same location on the gHgL dimer, implying that the two complexes are mutually exclusive (Chandramouli et al., 2017). This finding was confirmed by mass spectrometry (MS) and mutagenesis data, demonstrating that gL-
A B
in trimer (Ciferri et al., 2015a). The pentamer complex is hence required for viral entry in epithelial, endothelial and myeloid cells, and neuropilin-2 (Nrp2), was identified as the functional cell entry receptor (Martinez-Martin et al., 2018) (Figure 5). As the trimer, the pentamer showed additional high-affinity interactions with other host cell receptors, such as thrombomodulin (THBD), leukocyte immunoglobulin-like receptor subfamily B member 3 (LILRB3), immunoglobulin alpha Fc receptor (FCAR), and with lower affinity, CD46 (Stein et al., 2019). However, the biological relevance of this multitude of receptors remains to be understood. Sequence analysis of the pentamer subunits reveals an extremely high level of conservation, with a mean identity of 98%. This degree of conservation could be explained by the complex folding required to assemble the five subunits together (Chandramouli et al., 2017). Moreover, the multiple amino acid contacts occurring between the Nrp2 receptor and the gL, pUL128, pUL130, and pUL131A subunits also limit the possibility of antigenic drift (Martinez-Martin et al., 2018).
Figure 5. The HCMV gHgLpUL128/130/131A pentameric complex.
(A) Negative-stain electron microscopy and reference free 2D class averages of gHgLpUL128/130/131A in complex with 3G16 Fab and Nrp2. The image of gHgLpUL128/130/131A pentamer-Nrp2 complex revealed that only the pUL128 subunit interacts with Nrp2. (B) Random Conical Tilt (RCT) reconstruction of the gHgLpUL128/130/131A in complex with 3G16 Fab and Nrp2 receptor. From Martinez-Martin et al., 2018.
A B
1.2 Immune Control of HCMV Infection
HCMV has a complex interaction with the host with a long history of host/pathogen co- evolution that results in an infection with low morbidity and chronic infection instead of virus clearance. The control of HCMV infection requires the concerted activities of both innate and adaptive arms of the immune system (Figure 6) (Scalzo et al., 2007). The activation of the innate immune system is crucial in order to drive a high quality acquired immune response. Consequently, the establishment of a long-lasting immunity in response to a primary HCMV infection is required to control subsequent HCMV reactivation and prevent uncontrolled viral replication or serious HCMV diseases (Gerna et al., 2015; Smith et al., 2016). Within the adaptive response, both T cells and antibodies have been shown to be prominent. It is commonly believed that cellular immunity controls most HCMV replication. Nonetheless, HCMV-specific antibodies have been associated with the prevention and protection from reinfection as well as the congenital transmission of the virus (Bialas et al., 2016; Boppana and Britt, 1995; Itell et al., 2017). Despite this finding, it is clear that the humoral response on its own is not able to clear the virus and prevent viral reactivation (Collins-McMillen et al., 2018; Stern et al., 2019). The two arms of the adaptive immune response appear hence to be necessary for protection and resolution of HCMV infection (Krishna et al., 2019).
Figure 6. HCMV influences the immune system during the course of a lifetime.
Depicted are the three phases of HCMV infection: I) the acute/systemic phase, which is controlled after a few weeks; II) the persistent replication phase; and III) the latency phase, where the virus continues to shape and inflate host defenses. The control of HCMV infection requires the concerted activities of both innate and adaptive arms of the immune system. Modified from Picarda and Benedict 2018.
1.2.1 Innate Immune Response
The binding and entry of HCMV into the cell leads to the activation of a number of pathways resulting in the upregulation of the nuclear factor NFkB and interferon regulatory factor 3 (IRF3) which can ultimately lead to the production of primary interferon and inflammatory cytokines (Isaacson et al., 2008). This innate cellular response to the initial stages of HCMV infection is mediated by Toll like receptor 2 (TLR2) signaling, through the recognition of the viral surface glycoproteins gB and gH (Boehme et al., 2006; Compton et al., 2003).
Within the innate immune system, natural killer (NK) cells and mononuclear cells act as the first line of defense and play an important role in limiting early HCMV infection (Biron et al., 1999; Wilkinson et al., 2008). Human patients who lack or exhibit impaired NK cell function are highly susceptible to herpesvirus infections (Orange, 2013). Commensurate with their function as rapid responders, NK cells are primed to respond via the constitutive transcription of genes encoding cytokines, such as interferon-γ (IFNγ), and cytotoxic molecules (perforin and granzymes), thereby allowing the rapid secretion of these molecules on target cell engagement (Fehniger et al., 2007; Stetson et al., 2003). Although NK cells can be activated without the need for prior antigen exposure, they can also mediate antigen- specific viral infections (Cerwenka and Lanier, 2016). The innate immune response may shape or augment the adaptive immune response. The magnitude of the initial adaptive immune response is important in determining the numbers of antigen-specific memory T cells. During the transition from an innate immune response to adaptive anti-HCMV immunity, NK cells may be an important source of IFNγ, facilitating the expansion of antigen-specific helper T cells that are critical for HCMV control.
1.2.2 Cellular Immune Response
After NK cells trigger the release of inflammatory cytokines and cause lysis or apoptosis of infected cells, T cells restrict HCMV viral replication and viral spreading, although they are not able to definitively clear the virus (Boppana and Britt, 1996; Rist et al., 2005; Rohrlich et al., 2004). HCMV-specific T cells represent 10% of both CD4+ and CD8+ memory compartments in the peripheral blood of seropositive individuals (Sylwester et al., 2005).
CD8+ T cells release perforin and granzyme B to promote lysis of infected cells that present HCMV peptides on MHC-I (Gillespie et al., 2000; Hertoghs et al., 2010). On the other hand,
IFNγ and Tumor Necrosis Factor α (TNFα) (van Leeuwen et al., 2004). HCMV-specific T cells appears to be crucial for the control of primary viral infection and reactivation from latency (Reusser, 1991). In particular, CD8+ T cells were proven to have a protective role in both HSCT and SOT recipients (Greenberg et al., 1991; Lilleri et al., 2018; Reusser et al., 1992). Besides, both CD4+ and CD8+ T cell-mediated responses seems to be quite relevant in preventing HCMV congenital infection (Fornara et al., 2017; Huygens et al., 2014). The high frequencies of both CD4+ and CD8+ T cells associated with natural HCMV infection could result from virus reactivation and replication at epithelial sites, or reflect antigen presentation from viruses that persist in monocytes. Immune repertoire interrogation using 13,687 spanning peptides to cover 213 predicted HCMV-proteins (Kern et al., 2000) revealed that human T cells recognize at least 151 HCMV proteins. Among these, 81 proteins are recognized by both cell types, while CD4+ recognize 125 proteins and CD8+ recognize 107 proteins. Although the HCMV-specific T cell response is very broad, the vast majority points toward antigens that are highly conserved among different HCMV strains, such as the 65 kDa phosphoprotein (pp65), the IE1 proteins and the gB glycoprotein, which are recognized by more than 50% of seropositive individuals (Sylwester et al., 2005). One of the paradoxes of the cellular immunity against HCMV comes from the observation that HCMV does induce a very large T cell response, meanwhile the virus also possesses sophisticated and numerous immune evasion mechanisms (Reddehase et al., 2002). Among those, the capacity of the virus to enter a latency state has been the subject of intense investigation. It is known that HCMV establishes latency in the myeloid cell lineage and viral genome has been detected in peripheral blood monocytes and in CD34+ progenitors in the bone marrow (Hargett and Shenk, 2010; Mendelson et al., 1996; Taylor-Wiedeman et al., 1991). The viral gene expression program in these cells is restricted and extremely different from the one established during lytic infections. HCMV latent transcriptome is an area of extensive research, and at present, the exact roles of many latency-associated genes remain unclear (Goodrum et al., 2007; Reeves and Sinclair, 2010). T-cell repertoire interrogation identified four viral proteins expressed during latency called Latency Unique Natural Antigen (LUNA) (Bego et al., 2005), UL138 (Goodrum et al., 2007), US28 (Beisser et al., 2001), and viral IL-10 homologue (LAcmvIL-10) (Avdic et al., 2011). Interestingly, these proteins can be recognized by CD4+ T cells and T-cell responses specific for all four proteins are detectable in healthy HCMV-positive donors (Mason et al., 2013).
1.2.3 Humoral Immune Response
While T cells are thought to be crucial for the control of HCMV chronic infection (Klenerman and Oxenius, 2016), clinical data has provided evidence that humoral responses may offer protection against primary and acute infection, which may ultimately impact morbidity, transmission, and elimination of the virus. HCMV infection elicits a large quantity of antibodies targeting the most abundant viral proteins. These include structural tegument proteins (i.e., pp65), envelope glycoproteins such as gB, gM, gN, and the gHgL complexes (trimer and pentamer) as well as non-structural proteins (i.e., IE1) (Britt, 1991;
Landini and Michelson, 1988). Antibodies targeting antigens that are not exposed on the surface of the virion are unlikely to generate an efficient protection against the virus (Gomes et al., 2019). Nonetheless, antibodies targeting the envelope glycoproteins are able to neutralize viral infection in vitro and correlate with a decrease of viral transmission in primary infected pregnant women (Lilleri et al., 2013). Among these antibodies, those targeting the pentameric complex are the most potent neutralizers at least in vitro (Kabanova et al., 2014; Macagno et al., 2010). Anti-pULs-specific antibodies neutralize viral infection in epithelial cells at picomolar concentration, being thousand-fold superior than those targeting gHgL dimer or gB homotrimer (Macagno et al., 2010). The immunological rationale underlying this observation remains to be fully understood, since gHgL and gB glycoproteins together form the core machinery for viral membrane fusion (Vanarsdall et al., 2016). Indeed, monoclonal antibodies (mAbs) targeting the fusion machinery of other herpesvirus, such as EBV, are potent neutralizers (Bu et al., 2019). Immune repertoire interrogation of HCMV seropositive donors allowed the identification of multiple mAbs targeting the pULs subunits, demonstrating neutralizing activity conferred by blocking the molecular recognition of the pentamer to its cellular host receptor Nrp2 (Martinez-Martin et al., 2018). In contrast, neutralizing antibodies (nAbs) targeting gH are supposed to prevent the activation of the fusion machinery (Malito et al., 2018). The gB glycoprotein was shown to be the major target of humoral responses elicited towards HCMV following infection (Britt et al., 1990; Malito et al., 2018). Many human mAbs have been isolated from HCMV seropositive individuals and shown to neutralize infection in both fibroblasts and epithelial cells. Recently, two studies reported the results of the recombinant gB vaccine formulated in MF59 adjuvant, which demonstrated partial efficacy in reducing viremia after SOT and
al., 2018). These results might be explained by the finding that most antibodies induced by the vaccine lack a viral-neutralizing activity. Nonetheless, gB has been recently demonstrated to be the target of antibodies eliciting a strong antibody-dependent cellular phagocytosis (ADCP) in postpartum women and antibody-dependent cell mediated cytotoxicity (ADCC) after transplantation (Vietzen et al., 2020), making this HCMV glycoprotein an interesting target for vaccination.
1.2.4 Viral Immune Evasion
The large genome size owned by HCMV encodes 167 gene products, as well as non-coding RNAs and microRNAs, with an extensive alternate mRNA splicing. More than 40 HCMV gene products are recognized to have a role in modulating the host immune response following infection (Stern-Ginossar et al., 2009). HCMV evolved multiple mechanisms to evade the host immune response, allowing the virus to replicate and disseminate in the face of a competent innate and adaptive immune system. The mechanisms that modulate the infected cellular environment to limit immune recognition are most extensively expressed during lytic infection, but it is starting to become clear that viral gene activity during latency also acts to prevent immune clearance. During lytic infection, HCMV encodes for genes that directly modulate the innate/intrinsic immune responses (Amsler et al., 2013) or both intrinsic and extrinsic apoptosis pathways (Figure 7) (Fliss and Brune, 2012). Among the others, the tegument protein UL82 (pp71) evades antiviral immunity by inhibiting stimulator of interferon (STING) signaling (Fu et al., 2017), while UL145 facilitates degradation of the antiviral helicase like transcription factor (HLTF) by recruitment of Cullin4/DDB ligase complex (Nightingale et al., 2018). A number of HCMV-encoded genes expressed during lytic infection can interfere with both MHC class I and II restricted antigen processing and presentation. Proteins encoded within the US2-11 gene cluster target MHC class I and II molecules for retention within the endoplasmic reticulum (ER), re-direct MHC for degradation, and inhibit normal loading of peptides onto MHC class I (Gilbert et al., 1996; Jackson et al., 2011; Ploegh, 1998). Moreover, HCMV also exploits the unique strategy to undermine the host-viral immunity through the process known as molecular mimicry. The virus encodes proteins that are homologs of host immune-modulatory cytokines or their receptors, like UL111A that encodes cmvIL-10, an immunosuppressive IL-10 homolog (Avdic et al., 2011). The cmvIL-10 is a powerful inhibitor of Th1 cytokines
(such as IFNγ and IL-2) and, in addition, inhibits monocytes and macrophages inflammatory cytokines production resulting in a decreased surface expression of MHC class II with reduction of antigen presentation to CD4+ T cells. Another mechanism is MHC and peptide dependent mimicry, whereby the viral epitope mimics the host peptide ensuring the presentation of peptides without activation of immune effector cells (Banks and Rouse, 1992; Vider-Shalit et al., 2007). In addition, HCMV gene products UL18 and UL83 (pp65) encode for an MHC-I homolog inhibiting NK cell lysis (Patel et al., 2018). There is clearly a complex balance between virus immune evasion and host immune recognition, whose detailed and additional mechanisms have yet to be discovered.
Figure 7. HMCV modulation of the immune response.
Overview of the HCMV encoded genes that modulate the infected cellular environment to limit immune recognition. Red “T” bars indicate inhibition. Detail mechanisms are explained in the text.
Modified from Patro 2019.
1.3 Vaccines against HCMV
In the last decades, considerable efforts were deployed in the development of a HCMV vaccine that can prevent congenital infection in newborn, as well as primary infection or reactivation in immunocompromised patients. Given the severity of the disease induced by the virus, the US Institute of Medicine classified the generation of a vaccine against HCMV as a top priority since 1999 (Schleiss, 2008). Several HCMV vaccines have been evaluated in clinical trials, listed in Table 1 (Anderholm et al., 2016). These have included live- attenuated vaccines (i.e. Towne (Plotkin et al., 1994) and AD169 (Neff et al., 1979)), viral vectored vaccines expressing HCMV-encoded immunogens (i.e. alphavirus Venezuelan equine encephalitis (VEE) replicon particle (VRP) (Reap et al., 2007a; Reap et al., 2007b) and modified vaccinia virus Ankara (MVA) (Wang et al., 2006)), and purified recombinant vaccines co-administered with adjuvants. Among the latter, the most extensive studied vaccine to date is a subunit vaccine based on the viral envelope gB glycoprotein. However, none of the aforementioned candidates has been successful in achieving durable and protective immunity.
Table 1. HCMV vaccine categories evaluated in current and recent clinical trials.
Vaccine Category Phase Vaccine Antigens used
DNA/RNA-Based Vaccines
3 ASP0113 (Selinsky et al., 2005) pp65, gB 1 VCL-CT02 (Jacobson et al., 2009) pp65, gB, IE1
Vectored Vaccines
2 ALVAC-pp65 (Berencsi et al., 2001) pp65
1 ALVAC-gB (Adler et al., 1999) gB
1 AVX601 (Reap et al., 2007a; Reap et al., 2007b) gB, pp65, IE1 2 HCMV-MVA Triplex (Wang et al., 2006) pp65, IE1, IE2 Live-Attenuated
Viruses
2 Towne (Plotkin et al., 1994)
1 Towne-Toledo (Kemble et al., 1996)
Recombinant Subunit Vaccines
1 GSK1492903A (www.gsk-
clinicalstudyregister.com/study/108890#rs) gB
2 gB/MF59 (Pass et al., 1999) gB
VLP Vaccines 1 VBI-1501A (Kirchmeier et al., 2014) gB
1.3.1 Live-Attenuated Vaccines
The first efforts in the generation of a HCMV vaccine, were directed at developing a live, attenuated vaccine. The two main candidate vaccines in this class were the AD169 and Towne attenuated viruses (Neff et al., 1979; Plotkin et al., 1994), both derived from HCMV laboratory strains. The AD169 was found to be safe and well tolerated when administered in HCMV seronegative adults, however, this first vaccine was abandoned soon (Neff et al., 1979; Stern, 1984). On the other hand, Towne attenuated strain went on to extensive testing in SOT patients and healthy individual. Clinical trials demonstrated degree of protection comparable with that conferred by natural infection upon Towne vaccine administration (Brayman et al., 1988; Plotkin et al., 1994). Nonetheless, each study indicates that the vaccine failed to prevent HCMV infection. Towne was also assessed in a trial involving seronegative mothers who had children in daycare. Once again, the vaccine failed to protect women from HCMV infection transmitted from their children (Adler et al., 1995). The inability of Towne attenuated vaccine to convey protection in vaccinated patients might be due to over-attenuation of non-invasive vaccines, or genomic difference from clinical isolates. Indeed, although contributing to attenuation, the abrogation of the pentamer complex may have impaired the ability of the vaccinated host to develop antibody responses that would help block epithelial and endothelial infection following exposure to clinical strains, thereby potentially limiting the protective efficacy of immunization.
1.3.2 Recombinant gB Subunit Vaccines
While attenuated viruses were the first strategy investigated in HCMV vaccine development, subunit vaccines based on the recombinant gB have advanced the furthest in clinical trials. As mentioned beforehand, antibodies targeting gB are invariantly present in HCMV seropositive individuals, and importantly, were shown to hold virus neutralizing capacity (Britt et al., 1990; Marshall et al., 1992; Navarro et al., 1997). The recombinant gB vaccine developed, derived from the HCMV Towne strain gB sequence, has been modified such that the transmembrane domain and the furin cleavage site had been removed, and the cytoplasmic component has been fused with the truncated protein. Several phase II clinical trials utilizing the recombinant gB vaccine formulated in MF59 adjuvant (gB/MF59), an oil- in-water emulsion, have been completed (Bernstein et al., 2016; Griffiths et al., 2011; Pass,
immunogenic, eliciting an antibodies response superior than the one observed with placebo (Frey et al., 1999; Li et al., 2017). In addition, these studies showed a 50% efficacy in preventing primary HCMV infection in postpartum seronegative women (Pass et al., 2009), and reduction of viremia in SOT recipients (Griffiths et al., 2011). The protection of gB/MF59 vaccine was generally presumed, at least in the initial interpretation of studies results, to derive primarily from induction of virus-neutralizing antibody responses.
However, it was recently reported that although vaccination elicited strong gB-specific antibody response, minimal induction of HCMV nAbs was observed, and this despite the protection against infection/disease conferred. The gB glycoprotein has been recently demonstrated to be the target of antibodies eliciting a strong ADCP in postpartum women (Nelson et al., 2018) and ADCC after transplantation (Vietzen et al., 2020). Recently, a publication suggested that anti-AD2 serum titers correlate with protection from viremia (Baraniak et al., 2018b). Baraniak et al. demonstrated, however, that gB/MF59 vaccination only boosted AD2 responses in the 50% of HCMV+ individuals with a preexisting response and did not induce newly anti-AD2 antibodies in patients who lacked this response following natural infection. Most antibodies induced by the vaccine lack viral-neutralizing activity, thus being directed to decoy antigenic domains. Indeed, roughly 76% of the total anti-gB response was directed against AD3, diverting responses away from neutralization epitopes (Nelson et al., 2018). Similarly, the induction of non-neutralizing antibodies to other antigenic domains may promote antibody binding to gB that, in turn, may block access to more potent epitopes. A confirmation of this theory comes from recent analysis of an mRNA candidate vaccine encoding only the gB extracellular domain (without AD3) which elicit antibody responses with great durability and breadth than the MF59 adjuvanted gB protein (Nelson et al., 2020).
1.3.3 Viral Vectored Vaccines
A number of viral vectored HCMV vaccine have been developed, and some have been evaluated in phase I/II clinical studies. Generally, this vaccine technology utilizes viral vectors capable of human cell infection, without establishing a productive infection, expressing one or more HCMV antigens. In particular, two vaccines have been developed and tested using an attenuated Canarypox vector to deliver either gB (ALVAC-gB [vCP139]) or pp65 (ALVAC-pp65 [vCP260]) (Adler et al., 1999; Berencsi et al., 2001).
While gB-expressing ALVAC vaccine failed to increase neutralizing titers among seropositive recipients and did not induce significant nAbs in seronegative subjects, the ALVAC-pp65 has progressed to phase I clinical trials, showing a good safety profile as well as robust pp65-specific cytotoxic T lymphocytes (CTL) and antibody responses in seronegative adults (Heider et al., 2002). Another vectored platform, based on the alphavirus VEE, has progressed from animal models into clinical trials. In the HCMV vaccine AVX601, genes coding for VEE structural proteins were replaced with HCMV genes coding for the extracellular domain of gB and a pp65-IE1 fusion proteins in a double promoter replicon (Reap et al., 2007a; Reap et al., 2007b). As ALVAC-pp65, the alphavirus-based vectored vaccine was well tolerated and, upon immunization, seronegative recipients developed CTL and nAbs responses to all three HCMV antigens (Bernstein et al., 2009).
Recent studies have interrogated the immunogenicity of a MVA vaccine that expresses a variety of HCMV antigens, including gB, pp65, IE1, IE2 and the pentameric complex, in mice and nonhuman primate (NHP) models (Abel et al., 2011; Gillis et al., 2014; Wussow et al., 2014; Wussow et al., 2013). Interestingly, the MVA vector expressing all five of the protein components of the HCMV pentamer was shown to elicit a robust and durable neutralization titer that could prevent in vitro endothelial cells and Hofbauer macrophages (fetal cells present within placenta) infection (Wussow et al., 2014).
1.3.4 DNA/mRNA-Based Vaccines
Aside from the investigational vaccines mentioned above, DNA and mRNA vaccines are significant candidates. A plasmid-based DNA vaccine against HCMV has recently advanced to phase III clinical trials. The HCMV ASP00113 vaccine, developed by Vical, consists of two plasmids expressing pp65 (VCL-6368) and the extracellular domain of AD169 gB (VCL-6365) (Selinsky et al., 2005). Several preclinical studies in mice demonstrated that the administration of plasmid DNA lead to the expression of plasmid encoded antigen, production of antigen-specific antibodies, and protection against subsequent challenge with relevant pathogens (Smith et al., 2013; Tang et al., 1992; Ulmer et al., 1993). Consistently, ASP00113 vaccination of seronegative subjects elicited good gB antibody titers, as well as pp65 and gB specific T cell responses, whereas seropositive vaccinated individuals showed increases only in pp65-specific T cell responses (Wloch et al., 2008). In addition, in another trial it was demonstrated that administration of the DNA
vaccine significantly reduced viremia in HSCT recipients highlighting the efficacy of this vaccine strategy (Kharfan-Dabaja et al., 2012). In line with ASP00113, Vical developed another trivalent DNA vaccine (VCL-CT02), which includes the T-cell target IE1 in addition to the gB and pp65 coding sequences. Phase I clinical trial, showed that both cellular and humoral response to Towne vaccine were improved by the DNA vaccine priming (Jacobson et al., 2009). Nowadays, no RNA-based vaccine has been licensed for use in humans, despite promising results in preclinical studies. Indeed, a synthetic self- amplifying HCMV mRNA vaccine, containing gB and a pp65-IE1 fusion construct (Novartis), generated stunning CD4+ and CD8+ T-cell responses, as well as durable virus- neutralizing antibodies response in rhesus macaques (Brito et al., 2015; Geall et al., 2012), paving the way for this promising platform.
1.3.5 Virus-Like Particle Vaccines
Virus-like particles (VLP) are protein structures that resemble wild type viruses but do not have a viral genome nor infectious ability, creating in principle safer vaccine candidates.
Variation Biotechnologies vaccine Incorporated (VBI) laboratories developed a VLP vaccine expressing two gB variants: a gB-based VLP; and a chimeric gB-G protein, where the extracellular domain of the glycoprotein was membrane-anchored using the transmembrane and cytoplasmic domains of VSV, thought to maintain gB in a prefusion conformation (Kirchmeier et al., 2014). Both vaccines were found to induce neutralizing antibody titers 10-fold higher that the one induced with the same dose of soluble recombinant gB protein in mice. The vaccine is entered in phase I clinical trial enrolling HCMV-seronegative individuals for evaluation of safety and immunogenicity in early 2016 (Cui and Snapper, 2019). Another VLP HCMV vaccine candidate, expressing gB and pp65 antigens, has been developed by Redvax GmbH, a Swiss biopharmaceutical company. This technology is based on baculovirus-expressed proteins, instead of mammalian cells, minimizing the purification issues due to the relative large size of VLPs (Vicente et al., 2014). The Pfizer Vaccines Company has recently purchased this technology, aiming at a continuous development of the candidate HCMV vaccine platform.
1.4 Self-Assembling Nanoparticle for Antigen Display
One of the earliest examples of a self-assembling protein was reported in the 1970s, when the hepatitis B virus (HBV) surface antigen (HBsAg) was found to form spherical particles and, importantly, did not contain genetic material (Gerin et al., 1971). HBsAg-derive nanoparticle became the first efficacious HBV vaccine, licensed in 1981 (Hilleman, 2000), and represented a milestone that created a new pivot in the field of vaccinology (Figure 8).
It has long been known that an efficient immune response to a pathogen includes both antigen density and distribution of the latter on the surface of the microbe. Indeed, presenting multiple copies of an antigen in a repetitive array can drive more robust humoral immune responses than its soluble counterpart. This effect is thought to derive mainly from the multiple binding events occurring simultaneously between nanoparticle and host B cell receptors (BCRs) (Bachmann and Jennings, 2010). Among several technologies analyzed in this context (reviewed in (Gause et al., 2017; Irvine et al., 2015), self-assembling nanoparticles are at the frontline of multivalent antigen presentation (Lopez-Sagaseta et al., 2016). These carriers can form highly ordered, monodisperse structures that can be scalably manufactured and are naturally non-toxic, offering seamless integration of protein antigens via genetic fusion. A variety of self-assembling proteins, such as ferritin, lumazine synthase and de novo designed nanoparticle, have been successfully used as scaffolds to present complex antigens derived from influenza (Kanekiyo et al., 2013; Yassine et al., 2015), Human Immunodeficiency virus (HIV) (Abbott et al., 2018; He et al., 2016; Jardine et al., 2015), EBV (Kanekiyo et al., 2015) and Respiratory Syncytial Virus (RSV) (Marcandalli et al., 2019). Some of these functional self-assembling nanoparticles vaccine will be described below.
Figure 8. Self-assembly nanoparticles in vaccine development.
A timeline depicts the evolution of nanoparticles in vaccine development. Modified from Rappuoli and Serruto, 2019.
1.4.1 Ferritin-Based Nanoparticle
Ferritin is an important and ubiquitous iron-storage protein found in all living systems. It is expressed in most tissues, mainly as a cytosolic protein, but it can be also found at low concentration in the serum where it functions as an iron carrier (Theil, 1987). Ferritin is made of 24 subunits, each composed of a four-alpha-helix bundle (Figure 9A), that self- assemble in a quaternary structure with octahedral symmetry. Several high-resolution structures of ferritin have been determined, confirming that bacterial ferritin is made of 24 identical protomers (Cho et al., 2009), whereas in animals, ferritin is present as a protein complex, consisting of variable ratios of heavy and light subunits, assembling into particles of 24 subunits (Granier et al., 2003; Lawson et al., 1991). Ferritin self-assembles into nanoparticles, which present the advantage of being resistant to thermal and chemical stress (Pulsipher et al., 2017). Hence, Ferritin nanoparticle is potentially well suited to carry and expose immunogens. Moreover, Ferritin nanoparticles, self-arranging as an octahedron composed of eight trimeric units (Figure 9B), each with a 3-fold symmetry axis, allow the correct presentation of trimeric antigens at the nanoparticle surface (Figure 9C). Ferritin nanoparticle has been employed for the presentation of antigens from multiple viruses, such
as EBV (Kanekiyo et al., 2015), Hepatitits C virus (He et al., 2015), HIV (Jardine et al., 2013) and Influenza (Kanekiyo et al., 2013; Yassine et al., 2015). In all examples, nanoparticles yielded a very promising immune response in preclinical studies, as reflected by the notably higher number of neutralizing antibodies elicited. A development, and one of the major advantage of this carrier, is the possibility to display, on one nanoparticle, different antigenic proteins. In a recent work aiming towards a universal Influenza vaccine by providing broad coverage of protection against different virus strain, Kanekiyo and coworkers further developed and exploited the Ferritin nanoparticle system using a ferritin hybrid protein (Kanekiyo et al., 2019). The nanoparticle generated co-displayed the receptor-binding domain (RBD) of the last 90 years H1N1 influenza virus and showed that immunization of mice with this mosaic RBD-nanoparticle was capable of evoking a broad neutralizing antibody response covering all the known H1N1 strains. Moreover, a Ferritin nanoparticle vaccine carrying influenza and HIV-1 antigens on surface was shown to elicit broadly immune response against the selected viruses (Georgiev et al., 2018), confirming the newsworthy property of this carrier. Multiple display on Ferritin nanoparticle strengthened the possibilities for the generation of new vaccines against different pathogens or different antigens of a given pathogen.
Figure 9. Structure of Ferritin nanoparticle.
(A) Ferritin nanoparticles are composed of 24 subunits, each made of a four-alpha-helix bundle. The antigen of interest fuses to the N-terminus of the Ferritin polypeptide (shown in red). (B) Ferritin polypeptide self-assembles to form symmetry units with a 3-fold symmetry axis. (C) Fully assembled Ferritin nanoparticle, self-arranging as an octahedron composed of eight trimeric units.
Surface-exposed N-termini are shown in red. From Perotti and Perez, 2019.
1.4.2 De novo Design-Based Nanoparticle
Although many natural proteins have self-assembling properties (Goodsell and Olson, 2000), several groups have explored ways to design and produce nanoparticle materials based on de novo engineering. Structure-based design of nanoparticle immunogens has been limited by the restricted number of scaffolds available and the fact that their fundamental structural properties are fixed. Recent computational approaches improved the feasibility of designing self-assembling protein with atomic-level accuracy and customized structures (Bale et al., 2016; Hsia et al., 2016; King et al., 2014). Icosahedral point group symmetry, with trimeric protein scaffolds of known structure arranged with icosahedral symmetry, contains two- three- and five-fold axes of rotation. For instance, the I3-01 nanoparticle, exploited to display the HIV trimeric glycoprotein 41 (gp41) ectodomain on its surface, elicited mAb-expressing B cells more effectively than the soluble gp41 trimer (He et al., 2018; Hsia et al., 2016). The idea of combining proteins as building blocks into higher-order
nanoparticle (Drexler, 1981; Rajagopal and Schneider, 2004). Recently, Marcandalli et al., have designed a self-assembling protein nanoparticle displaying a fused variant of the stabilized pre-fusion glycoprotein (DS-Cav1) of RSV. The I53-50 nanoparticle scaffold, made up of two components with icosahedral symmetry, enabled the production of immunogens that displayed 20 copies of the trimeric DS-Cav1 in a symmetrical array on the exterior surface of the nanoparticle (Figure 10) (Marcandalli et al., 2019). The I53-50 new vaccine candidate induced a 10-fold gain in neutralizing antibodies achieved due to structure-based stabilization of the antigen and fusion to the nanoparticle. In another additional study, native-like HIV-1 envelope trimers were displayed in a multivalent fashion on the I53-50 nanoparticle scaffold and, once again, immunization studies revealed stronger effective priming compared to the soluble antigen (Brouwer et al., 2019). Given the rapidly expanding capabilities of computational protein design (Huang et al., 2016), continued development of structure-based self-assembling immunogens will surely have an impact on next generation vaccines.
Figure 10. Self-assembly of the I53-50 nanoparticle.
(A) The polypeptide I53–50A.1NT1 naturally trimerizes forming a 3-fold symmetry axis. The N- terminus projecting outward, where antigens can be displayed, is shown in red. (B) The second component of the I53-50 nanoparticle is the I53–50B.4PT1 protein (here shown in orange) that assembles into a pentamer. (C) I53–50A.1NT1 trimer and I53–50B.4PT1 pentamer self-assemble as a nanoparticle when put in contact. (D) The fully assembled I53-50 nanoparticles are formed by 20 trimers and 12 pentamers. The N-termini of I53–50A.1NT1 is shown in red, and the black triangle represents the 3-fold symmetry axis of each pair of trimers. From Perotti and Perez, 2019.
2 Aim of the Thesis
The human Cytomegalovirus (HCMV) is a Beta-herpesvirus that, after primary infection, establishes a lifelong persistence in the human host. HCMV infection is usually asymptomatic; however, the virus is responsible for high morbidity and mortality in immunocompromised patients and remains the leading viral cause of congenital birth defects. Despite decades of research dedicated to the biology of this herpesvirus, there are currently no vaccine or treatment options available. Among the HCMV surface glycoproteins (g), gB-specific antibodies are able to block infection in all cell types, and account for the majority of neutralizing antibodies (nAbs) activity in the sera of infected humans. The gB protein has been the focus of many vaccine efforts, which, surprisingly, failed to elicit an adequate nAbs titer and, therefore, reach full protection.
Faced with challenging pathogens, modern vaccinology exploits different techniques to achieve its goal. Some of these include antibody-guided antigen discovery, engineering of antigens to increase the stability or modification to express particular domains or epitopes. Furthermore, multivalent antigen presentation, proven by self- assembling proteins, further increases the induction of nAbs, improving the likelihood of protective efficacy in humans. In this work, we set out to overcome empirical approaches and generate a new HCMV vaccine with an improved immunogenicity. In particular, we combined our breakthroughs in gB antigen immunogenicity and custom protein nanomaterial design to produce two nanoparticle immunogens that induce up to 100-fold more potent neutralizing responses than the most studied recombinant gB vaccine.
These data were submitted for publication (see 3):
Rationally Designed Human Cytomegalovirus gB Nanoparticle Vaccine with Improved Immunogenicity. Michela Perotti*, Jessica Marcandalli*, Davide Demurtas, Federica Sallusto, Laurent Perez. *equal contribution
3 Results
Rationally Designed Human Cytomegalovirus gB Nanoparticle Vaccine with Improved Immunogenicity.
Authors: Michela Perotti1,2,*, Jessica Marcandalli1,3,*, Davide Demurtas4, Federica Sallusto1,2 and Laurent Perez1,5,#. *equal contribution
1 Institute for Research in Biomedicine, Università della Svizzera italiana, faculty of Biomedical Sciences, 6500 Bellinzona, Switzerland.
2 Institute of Microbiology, ETH Zürich, 8093 Zürich, Switzerland
3 Humabs BioMed SA, Vir Biotechnology, Bellinzona, Switzerland
4 BioEM Facility, School of Life Sciences, Swiss Federal Institute of Technology Lausanne (EPFL), Lausanne, Switzerland
5 Service of Immunology and Allergy, Department of Medicine, Lausanne University Hospital, University of Lausanne, Lausanne, Switzerland.
# Corresponding author: laurent.perez@unil.ch
Submitted for publication
My contribution:
Figure 1: HCMV gB antigens design and biochemical analysis.
Figure 2: Serum binding and neutralizing titers in mice immunized with gB antigens.
Figure 3D: Reducing SDS-PAGE of SEC-purified components and nanoparticle immunogens.
Figure 4: Serum binding and neutralizing titers in mice immunized with nanoparticle immunogens.
S.Figure 2: Serum binding and neutralizing titers with complement addition in mice immunized with gB antigens.
S.Figure 4: Flow cytometry detection of gBfull-length in Expi293 transfected cells
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Rationally designed Human Cytomegalovirus gB nanoparticle
1
vaccine with improved immunogenicity
2 3
Michela Perotti 1,2,6, Jessica Marcandalli1,3,6 , Davide Demurtas4, Federica Sallusto1,2 and Laurent 4
Perez 1,5,7,*
5 6
1Institute for Research in Biomedicine, Università della Svizzera italiana, faculty of Biomedical 7
Sciences, 6500 Bellinzona, Switzerland.
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2Institute of Microbiology, ETH Zürich, 8093 Zürich, Switzerland 10
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3Humabs BioMed SA, Vir Biotechnology, Bellinzona, Switzerland 12
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4BioEM Facility, School of Life Sciences, Swiss Federal Institute of Technology Lausanne 14
(EPFL), Lausanne, Switzerland 15
16
5Service of Immunology and Allergy, Department of Medicine, Lausanne University Hospital, 17
University of Lausanne, Lausanne, Switzerland.
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6These authors contributed equally 20
21
7Lead Contact 22
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*Correspondence: laurent.perez@unil.ch 24
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Keywords: Human Cytomegalovirus; glycoprotein B; nanoparticle; vaccine 27
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Combined Manuscript File
