Chikungunya virus-like particle vaccine

S.W.H. Metz

Research output: Thesisinternal PhD, WU

Abstract

 

Chikungunya virus (CHIKV) is an arthropod-borne alphavirus (family Togaviridae) and is the causative agent of chikungunya fever. This disease is characterised by the sudden onset of high fever and long-lasting arthritic disease. First identified in Tanzania in 1952, CHIKV has re-emerged in the last decade causing large outbreaks throughout Africa, Asia and Southern Europe. Increased CHIKV spread is mainly caused by its adaptation to a new mosquito vector, the Asian tiger mosquito Ae. albopictus, which is able to colonize more temperate regions. Currently, there are no antiviral treatments or commercial vaccines available, to prevent CHIKV infections. However, increased vector spread and clinical manifestations in humans, have triggered vaccine development. A broad range of vaccine strategies have been proposed and described, including inactivated virus formulations, live-attenuated virus, chimeric virus vaccines, DNA vaccines, adenoviral vectored vaccines, subunit protein vaccines and virus-like particle (VLP) formulations. However, these vaccination strategies have specific limitations in manufacturing, immunogenicity, safety, recombination and large scale production. Many, if not all safety problems do not apply for subunit or VLP based vaccines, except for the recombinant origin of the vaccine.

Recently, a CHIKV VLP-based vaccine was developed and provided protection in both mice and non-human primates. Even though this VLP approach is a safe, efficient and promising alternative to other vaccine strategies, large scale DNA plasmid transfection into mammalian cells and VLP yield of transfected cells remains challenging in terms of industrial production. These problems are alleviated by using the recombinant baculovirus-insect cell expression system.

In this thesis, recombinant baculoviruses were constructed to produce CHIKV glycoprotein E1 and E2 subunits and VLPs.For the production of CHIKV-E1 and E2 subunits, both protein genes were cloned downstream the polyhedrin gene (polh) promoter of in an Autographica californica multiple nucleopolyhedrovirus backbone, together with their authentic signal peptides 6K(E1) and E3(E2). Deletion of the C-terminal transmembrane domain, generated secreted versions of E1 (E1ΔTM or sE1) and E2 (E2ΔTM or sE2). A substantial amount of recombinant protein was glycosylated and processed by furin. The secreted CHIKV subunits were purified from the medium and were able to induce neutralizing antibodies in rabbits. For the production of the VLPs, the complete structural polyprotein (capsid, E3, E2, 6K, E1) was cloned downstream the AcMNPV polh promoter. E3E2 precursor processing and glycosylation appeared to be more efficient when E3E2 were expressed as part of the whole structural polyprotein cassette, compared to the individually expressed E3E2. The VLPs were isolated from the medium fraction and were morphologically similar to wild type CHIKV. A similar strategy was used to produce VLPs from another alphavirus, the salmonid alphavirus (SAV). Here, however, the normal baculovirus expression temperature of 27°C appeared to be detrimental for SAV-E3E2 furin cleavage and SAV-VLP production. E2-glycoprotein processing was shown to be temperature dependent and a tailored temperature-shift regime was designed in which Sf9-cells were infected with a recombinant baculovirus expressing the SAV structural proteins, and incubated at 27°C for 24 h, followed by a processing phase of 72 h at 15°C. Using this temperature regime, SAV-VLPs were produced that were morphologically indistinguishable from wild type SAV and underscores the flexibility of the baculovirus-insect cell expression system.

The immunogenicity of purified CHIKV-sE1 and -sE2 subunits and purified CHIKV-VLPs were then tested in a lethal vaccination-challenge mouse model, in IFN α/β, -γ receptor null AG129 mice. The innate immune system of these mice was made dysfunctional. This vaccine-challenge study clearly showed that VLPs provided superior protection, compared to their subunit counterparts. The subunits provided only partial protection and induced low neutralizing antibody titres. Immunization with the VLPs fully protected mice against lethal challenge and induced significant higher neutralizing antibody titress. Even though neutralizing antibody titres were lower after subunit immunization, this study showed that a minor neutralizing antibody response is sufficient to protect mice from lethal CHIKV challenge. Next, the CHIKV VLPs were tested for their ability to induce complete protection in an adult wild-type immune-competent mouse model, in which mice develop arthritic disease after CHIKV infection. The VLPs were able to induce full protection after a single immunization of 1 µg VLPs, without the use of adjuvants. In addition, IgG isotyping revealed a balanced IgG1-IgG2c response, suggesting a role for both humoral and cellular immunity in the protection against CHIKV infection. Mice served as a proxy for primates and vaccination trials in primates are next on the agenda.

This thesis is a typical example of the opportunities for the recombinant baculovirus-insect cell expression system in viral vaccine development, especially in vaccine development for other arboviruses. Although the CHIKV-VLPs produced in insect cells are amenable for large-scale production, the production process and downstream processing need to be carefully designed and optimized before CHIKV VLPs can be produced on an industrial scale. However, the data presented in this thesis show that CHIKV-VLPs produced in insect cells using recombinant baculoviruses represents as a new, safe, non-replicating and effective vaccine candidate against CHIKV infections.

Chikungunya virus (CHIKV) is an arthropod-borne alphavirus (family Togaviridae) and is the causative agent of chikungunya fever. This disease is characterised by the sudden onset of high fever and long-lasting arthritic disease. First identified in Tanzania in 1952, CHIKV has re-emerged in the last decade causing large outbreaks throughout Africa, Asia and Southern Europe. Increased CHIKV spread is mainly caused by its adaptation to a new mosquito vector, the Asian tiger mosquito Ae. albopictus, which is able to colonize more temperate regions. Currently, there are no antiviral treatments or commercial vaccines available, to prevent CHIKV infections. However, increased vector spread and clinical manifestations in humans, have triggered vaccine development. A broad range of vaccine strategies have been proposed and described, including inactivated virus formulations, live-attenuated virus, chimeric virus vaccines, DNA vaccines, adenoviral vectored vaccines, subunit protein vaccines and virus-like particle (VLP) formulations. However, these vaccination strategies have specific limitations in manufacturing, immunogenicity, safety, recombination and large scale production. Many, if not all safety problems do not apply for subunit or VLP based vaccines, except for the recombinant origin of the vaccine.

Recently, a CHIKV VLP-based vaccine was developed and provided protection in both mice and non-human primates. Even though this VLP approach is a safe, efficient and promising alternative to other vaccine strategies, large scale DNA plasmid transfection into mammalian cells and VLP yield of transfected cells remains challenging in terms of industrial production. These problems are alleviated by using the recombinant baculovirus-insect cell expression system.

In this thesis, recombinant baculoviruses were constructed to produce CHIKV glycoprotein E1 and E2 subunits and VLPs.For the production of CHIKV-E1 and E2 subunits, both protein genes were cloned downstream the polyhedrin gene (polh) promoter of in an Autographica californica multiple nucleopolyhedrovirus backbone, together with their authentic signal peptides 6K(E1) and E3(E2). Deletion of the C-terminal transmembrane domain, generated secreted versions of E1 (E1ΔTM or sE1) and E2 (E2ΔTM or sE2). A substantial amount of recombinant protein was glycosylated and processed by furin. The secreted CHIKV subunits were purified from the medium and were able to induce neutralizing antibodies in rabbits. For the production of the VLPs, the complete structural polyprotein (capsid, E3, E2, 6K, E1) was cloned downstream the AcMNPV polh promoter. E3E2 precursor processing and glycosylation appeared to be more efficient when E3E2 were expressed as part of the whole structural polyprotein cassette, compared to the individually expressed E3E2. The VLPs were isolated from the medium fraction and were morphologically similar to wild type CHIKV. A similar strategy was used to produce VLPs from another alphavirus, the salmonid alphavirus (SAV). Here, however, the normal baculovirus expression temperature of 27°C appeared to be detrimental for SAV-E3E2 furin cleavage and SAV-VLP production. E2-glycoprotein processing was shown to be temperature dependent and a tailored temperature-shift regime was designed in which Sf9-cells were infected with a recombinant baculovirus expressing the SAV structural proteins, and incubated at 27°C for 24 h, followed by a processing phase of 72 h at 15°C. Using this temperature regime, SAV-VLPs were produced that were morphologically indistinguishable from wild type SAV and underscores the flexibility of the baculovirus-insect cell expression system.

The immunogenicity of purified CHIKV-sE1 and -sE2 subunits and purified CHIKV-VLPs were then tested in a lethal vaccination-challenge mouse model, in IFN α/β, -γ receptor null AG129 mice. The innate immune system of these mice was made dysfunctional. This vaccine-challenge study clearly showed that VLPs provided superior protection, compared to their subunit counterparts. The subunits provided only partial protection and induced low neutralizing antibody titres. Immunization with the VLPs fully protected mice against lethal challenge and induced significant higher neutralizing antibody titress. Even though neutralizing antibody titres were lower after subunit immunization, this study showed that a minor neutralizing antibody response is sufficient to protect mice from lethal CHIKV challenge. Next, the CHIKV VLPs were tested for their ability to induce complete protection in an adult wild-type immune-competent mouse model, in which mice develop arthritic disease after CHIKV infection. The VLPs were able to induce full protection after a single immunization of 1 µg VLPs, without the use of adjuvants. In addition, IgG isotyping revealed a balanced IgG1-IgG2c response, suggesting a role for both humoral and cellular immunity in the protection against CHIKV infection. Mice served as a proxy for primates and vaccination trials in primates are next on the agenda.

This thesis is a typical example of the opportunities for the recombinant baculovirus-insect cell expression system in viral vaccine development, especially in vaccine development for other arboviruses. Although the CHIKV-VLPs produced in insect cells are amenable for large-scale production, the production process and downstream processing need to be carefully designed and optimized before CHIKV VLPs can be produced on an industrial scale. However, the data presented in this thesis show that CHIKV-VLPs produced in insect cells using recombinant baculoviruses represents as a new, safe, non-replicating and effective vaccine candidate against CHIKV infections.

Original languageEnglish
QualificationDoctor of Philosophy
Awarding Institution
  • Wageningen University
Supervisors/Advisors
  • Vlak, Just, Promotor
  • Pijlman, Gorben, Co-promotor
Award date24 Jun 2013
Place of PublicationS.L.
Publisher
Print ISBNs9789461735652
Publication statusPublished - 2013

Keywords

  • chikungunya virus
  • virus-like particles
  • viral diseases
  • aedes albopictus
  • disease vectors
  • vaccines
  • vaccine development
  • gene expression
  • baculovirus
  • insects
  • cell culture vaccines

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    Cite this

    Metz, S. W. H. (2013). Chikungunya virus-like particle vaccine. S.L.: s.n.