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Therapeutic potential of neutralizing antibodies
Despite the recent advances in HCV treatment, vaccination would still represent the best way to reduce the global burden of HCV. However, to date no licensed vaccine exists against this virus. Most of the successful vaccines are based primarily on the induction of potent nAb responses (Lambert et al., 2005) and to date a number of studies have demonstrated that nAbs are capable of controlling HCV infection (as described above). This is encouraging for the potential development of at least a partially anti-HCV vaccine ideally capable of inducing long-term B-cell and T-cell memory responses. Unfortunately, a number of difficulties, such as the emergence of neutralization escape variants, HCV cell-to-cell transmission, and the ability of HCV to re-infect previously exposed persons, makes the development of an efficient HCV vaccine a major challenge.
The traditional approach of using inactivated viruses for vaccination against HCV has received little attention due to the difficulties in producing large quantities of infectious HCV particles in cell culture. Akazawa et al., who used the inactivated genotype 2 (J6/JFH-1) HCVcc to immunize mice, demonstrated the feasibility of an inactivated whole virus vaccine.
Induced NAbs were able to neutralize genotypes 1a, 1b and 2a in vitro and could protect human liver chimeric uPA-SCID mice from experimental challenge with the lowest virus dose. Interestingly, immunization of mice with inactivated HCVcc resulted in more efficiently neutralizing serum than vaccination with recombinant E1 and E2, implying that HCV particles may be more immunogenic than just the individual recombinant envelope proteins. In addition, another study demonstrated that VLPs pseudotyped with HCV envelope glycoproteins induced high titer of bnAbs in mice and macaques (Garrone et al., 2011).
The only anti-HCV vaccine tested in humans was developed by Chiron Corporation (now Novartis, Basel, Switzerland). It used as immunogen a heterodimer of recombinant E1 and E2 produced in mammalian cells. In initial trials this vaccine induced an anti-E2 antibody response in chimpanzees, which was protective against challenge with homologous virus (Choo et al., 1994). However, the challenge with a heterologous virus strain protected only one chimpanzee of nine, indicating that in most vaccinated animals the induced immune response was not sterilizing (Houghton & Abrignani, 2005). A recombinant vaccine containing genotype 1a glycoproteins E1/E2 combined with an oil–water adjuvant has also been assessed in human volunteers. The initial results revealed that approximately half of the vaccinated people had antibodies against HVR1 and some also possessed antibodies against epitope I, and epitope II as well as the E1 region 313-327. Moreover, vaccinees did not experience any significant adverse events (Frey et al., 2010; Ray et al., 2010). In a follow-up study, the serum of one out of sixteen persons vaccinated with a single HCV strain of genotype 1a possessed cross-neutralizing activity of all seven HCV genotypes (Law et al., 2013). These results prove that bnAbs can be induced by recombinant E1/E2 vaccines at least in some individuals. However, a number of difficulties associated with the development of an efficient vaccine, such as relatively low titers of bnAbs and average levels of neutralization, need to be addressed. One problem is that in the case of immunization with unmodified E2 or E1E2, HVR1 is immunodominant, implying that engineered immunogens lacking HVR1 may be needed. Finally, due to the multiple escape mechanisms of HCV from the immune response, targeting a single viral
component is not likely to be sufficient. As a result, vaccine development efforts should focus on designing immunogens that are capable of inducing nAbs targeting several epitopes on the envelope proteins. In addition, it would be interesting to better analyze the nature and specificity of nAbs during the acute phase of self-resolving HCV infection, since those antibodies are likely to have the highest protective potency. One study has revealed the higher prevalence of a mAbs recognizing discontinuous epitopes (E1 (aa 297-306)-E2 (aa 480-494)-E2 (aa613-621)) in patients who spontaneously cleared infection (Ndongo et al., 2010).
Infection inhibition by the recombinant GBV-B E2e
A number of different constructs of the recombinant GBVB E2 ectodomain (E2e) were produced during the study. However, all of the mAbs we obtained from the immunization of mice with the recombinant GBV-B E2e appeared to recognize linear epitopes, as they reacted with E2 in Western blot, thereby making it difficult to verify the correct conformation of our recombinant GBV-B E2e constructs. Such verification can be done by measuring the recombinant GBV-B E2e ability to inhibit infection of primary hepatocytes from marmosets, which likely occurs by competition with infectious GB virus B for entry receptors. We therefore initiated a collaboration with the laboratory of Annette Martin at Institut Pasteur, who studies GBV-B entry and replication. We tried to inhibit infection with GBV-B E2350-581, which represents the full-length ectodomain of GBV-B E2, and a shorter variant of GBV-B ectodomain, E2350-540 (Figure 14).
Crystallization trials of GBV-B E2e with antibody fragments
Multi-domain, heavily glycosylated viral envelope proteins are often difficult to crystallize, and different strategies can be applied to achieve crystallization in these cases. One of these strategies is the use of crystallization “chaperones”, which are ligands that promote the formation of a crystal lattice when assayed in complex with the protein being studied. We decided to use this strategy and so, to obtain specific ligands for GBV-B E2e to perform co-crystallization experiments.
For this purpose, we immunized mice with the recombinant ectodomain and obtained a number of monoclonal antibodies (mAbs) specifically recognizing the glycoprotein. The main characteristics of these mAbs are summarized in Table 2. I determined the antibody sequence from the hybridoma cDNA and cloned the variable domains into a plasmid for production of recombinant antibody fragments (Fabs and single chain variable fragments (scFvs)) in Drosophila S2 cells. This enabled me to produce and purify large quantities of these ligands. Competition analysis of those antibody fragments by SEC revealed that three (E19.4, F7.7 and F16.1) out of five mAbs cross-compete for binding to the same antigenic region on GBVB E2 (Figure 18). The mAb F24.3 had a lower affinity for GBV-B E2e, rendering it more difficult to determine cross-competition patterns with other antibodies by SEC. Two of the mAbs (C23.21 and D18.6) were found to bind the affinity purification tag (double Strep-tag® II) (http://www.iba-lifesciences.com/strep-tag.html), which had not been proteolytically removed from the protein used for immunizations. A stable monoclonal HEK293T cell line expressing the Fab derived from C23.21 mAb was established, enabling the production of this ligand, and which is used for very specific recognition of the Strep-Tag on many different proteins.
Crystallization of deglycosylated GBV-B E2!C
Although GBV-B E2 is less extensively glycosylated than its HCV counterpart (6 predicted N-linked glycans in GBV-B E2 instead of 11 in HCV E2), the glycans present may hinder crystallization of the protein given the high degree of flexibility of the sugar chains. One way of avoiding this problem is enzymatic deglycosylation of the protein after purification from the supernatant. This is preferable to mutating the N-glycosylation sites since glycosylation may be important during folding in the endoplasmic reticulum. In addition, the Strep tag, which is fused at the C-terminal end of the recombinant glycoprotein as well as to the Fab C-terminal end, may also interfere with crystallization. Therefore, I developed a protocol for deglycosylation and the enzymatic removal of the affinity tag from GBV-B E2ΔC and the Fabs. The double Strep affinity tag was removed from E2ΔC and the Fab by specific proteolytic cleavage with EKMax Enterokinase (Invitrogen, San Diego, USA). The amount of enterokinase required to achieve complete removal of the tag was optimized for each protein in a small-scale reaction (Figure 24). The reaction was then scaled up in a linear manner and the protein without the Strep-tag was purified from the reaction mixture.
Table of contents :
Acknowledgements
Table of Contents
List.of.abbreviations
General Introduction
Virus.Entry
The role of viral envelope proteins in virus entry
Virus interaction with cell surface molecules
Principles of viral membrane fusion
Mechanisms!to!trigger viral membrane fusion
Structures!of!viral!fusion!proteins!
The!role!of!viral!envelope!proteins!in!immune!evasion!
Chapter.I Hepaciviruses.and.their.entry.to.target.cells
Introduction
Hepatitis C Virus
HCV pathogenesis
Animal models!for!HCV
GBVGB!as a surrogate!model!for!HCV
Genome organization of Hepaciviruses
Structural proteins
NonGstructural proteins
HCV genotypes
Current treatment options
Experimental systems for studying!HCV and GBVGB entry in,vitro
Infectious cycle!of HCV and GBVGB
Role of neutralizing antibodies in HCV infection
HCV immune escape strategies
3D structures of the main neutralizing epitopes
The rapeutic potential of neutralizing antibodies
Immune responses to GBVGB virus
Structural Characterization of the GB virus B Envelope Glycoprotein.E2
Background
Objectives
Results
Discussion!
Materials!and!methods!
II..The.Structure.Of.the.Hepatitis.C.Virus.Envelope.Glycoprotein.E2.Antigenic.Site.529N
540.in.Complex.With.Antibody.DAO5
Background!
Objective!
Results!
Discussion!
Materials!and!Methods!
Chapter.II Baculovirus Envelope Protein F
Introduction
Baculoviridae family
Replication cycle
Baculovirus envelope fusion proteins
Characteristics of baculovirus F protein
The structural similarity between baculovirus F and paramyxovirus F proteins
ReceptorGbinding!function!of!baculovirus F
Relationship between insect retroviruses and baculoviruses
Cellular!orthologs!of!baculovirus!F!protein!
Structural characterization of a baculovirus fusion protein ectodomain
Background
Objectives
Results
Discussion
Materials and Methods
Final.Discussion
Supplementary.Materials.and.Methods
References
Appendix
Crystallography techniques and terms used in the thesis
Table.of.Figures
Table.of.Tables.
