A VLP-based vaccine targeting domain III of the West Nile virus E protein protects from lethal infection in mice
© Spohn et al. 2010
Received: 21 April 2010
Accepted: 6 July 2010
Published: 6 July 2010
Since its first appearance in the USA in 1999, West Nile virus (WNV) has spread in the Western hemisphere and continues to represent an important public health concern. In the absence of effective treatment, there is a medical need for the development of a safe and efficient vaccine. Live attenuated WNV vaccines have shown promise in preclinical and clinical studies but might carry inherent risks due to the possibility of reversion to more virulent forms. Subunit vaccines based on the large envelope (E) glycoprotein of WNV have therefore been explored as an alternative approach. Although these vaccines were shown to protect from disease in animal models, multiple injections and/or strong adjuvants were required to reach efficacy, underscoring the need for more immunogenic, yet safe DIII-based vaccines.
We produced a conjugate vaccine against WNV consisting of recombinantly expressed domain III (DIII) of the E glycoprotein chemically cross-linked to virus-like particles derived from the recently discovered bacteriophage AP205. In contrast to isolated DIII protein, which required three administrations to induce detectable antibody titers in mice, high titers of DIII-specific antibodies were induced after a single injection of the conjugate vaccine. These antibodies were able to neutralize the virus in vitro and provided partial protection from a challenge with a lethal dose of WNV. Three injections of the vaccine induced high titers of virus-neutralizing antibodies, and completely protected mice from WNV infection.
The immunogenicity of DIII can be strongly enhanced by conjugation to virus-like particles of the bacteriophage AP205. The superior immunogenicity of the conjugate vaccine with respect to other DIII-based subunit vaccines, its anticipated favourable safety profile and low production costs highlight its potential as an efficacious and cost-effective prophylaxis against WNV.
West Nile virus (WNV) is a positive-stranded RNA flavivirus grouped within the Japanese encephalitis virus serocomplex. Transmitted primarily between birds via Culex mosquitoes, it occasionally infects humans, where it usually remains asymptomatic or causes a mild undifferentiated febrile illness called West Nile fever. Under certain conditions, mainly in immunocompromised or elderly individuals, and in individuals deficient in expression of the chemokine receptor CCR5, WNV infection can develop into severe, potentially life-threatening encephalitis [1–4]. In 2002, WNV was responsible for the largest outbreak of arthropod-borne encephalitis recorded in the USA, accounting for 2946 diagnosed cases and 284 deaths . Since then the virus has been spreading throughout the USA, as well as Canada, Mexico and the Caribbean basin . Isolated clinical cases have also been reported in recent years in Mediterranean countries, suggesting emergence of the virus in Western Europe [7, 8]. In the absence of an effective treatment, there is a medical need for the development of a safe and efficient prophylactic vaccine against WNV.
A chimeric virus incorporating the envelope proteins of WNV into the infectious backbone of a yellow fever vaccine strain is currently being developed as a live-attenuated vaccine [9–11]. While immunogenic in humans, such a vaccine carries the inherent risk of reversion to a more virulent form, requiring stringent monitoring of the production process and careful safety assessment during clinical development. Alternative vaccination strategies are therefore focusing on recombinant subunit vaccines based on the large envelope glycoprotein (E) of WNV. The E protein is crucial for virus attachment and entry into host cells and is also the major antigen eliciting neutralizing antibody responses . In particular a structurally distinct domain of the E protein (DIII) has been proposed as the receptor-binding domain . Antibodies recognizing epitopes in this domain have been shown to neutralize the virus in vitro [14–19] and passive transfer of DIII-specific antibodies has been shown to protect mice from WNV challenge . Subunit vaccines based on recombinantly expressed DIII have been tested in animal models and have proven effective in protecting from WNV infection [20–24]. However, multiple injections and/or strong adjuvants were needed to induce neutralizing antibody responses, indicating that isolated DIII is poorly immunogenic.
We have previously shown that by displaying antigens in a repetitive and highly ordered fashion on the surface of virus-like particles (VLPs) derived from the bacteriophage Qβ, specific B cells can be efficiently activated and rapid and robust antibody responses can be induced [25–28]. Here we describe the production of a conjugate vaccine based on recombinant DIII covalently linked to VLPs derived from the recently discovered bacteriophage AP205. A single injection of the conjugate vaccine was sufficient to induce virus-neutralizing antibodies and provide significant protection from WNV challenge, demonstrating its superior immunogenicity over previously described DIII-based vaccines and highlighting its potential as an efficient and safe prophylaxis against WNV.
Production of the AP205 VLP Carrier
Production of the DIII-C-AP205 Conjugate Vaccine
Immunogenicity of DIII-C-AP205 in Mice
Vaccination with DIII-C-AP205 Induces Neutralizing Antibodies and Protects from Lethal WNV Infection
Domain III of the envelope protein of WNV is a major target of virus-neutralizing antibody responses and has been identified as a promising candidate antigen for the development of recombinant subunit vaccines . In this study we produced a highly immunogenic and efficacious WNV vaccine consisting of recombinant domain III chemically cross-linked to virus-like particles of the bacteriophage AP205. The conjugate vaccine produced high DIII-specific antibody titers in mice, which were able to efficiently inhibit viral replication both in vitro and in vivo. In contrast to other experimental vaccines based on recombinant DIII and comprising different adjuvants [20–23], a single injection in the presence of Alum was sufficient to induce neutralizing antibody responses and confer partial protection from WNV challenge (Figure 4). Interestingly, the neutralizing titers observed (approximately 30-40) were in the range of those induced by a single injection of live WNV vaccines such as a chimeric attenuated flavivirus vaccine [9, 35] or a recombinant attenuated influenza strain expressing the WNV E protein . Multiple injections either in the presence or absence of Alum as adjuvant yielded sustained high titers of DIII-specific antibodies, which efficiently neutralized the virus. The reason for the superior immunogenicity of the DIII-C-AP205 conjugate vaccine most likely resides in its virus-like connotations. It has been shown that highly ordered and repetitive antigen arrays can cause an efficient cross-linking of BCRs on specific B cells and induce a rapid and sustained antibody response . As domain III is presented to the immune system in an oriented and densely packaged fashion on the surface of the AP205 VLP, it is likely that DIII-specific B cells are promptly and efficiently activated to produce specific IgG antibodies. The particulate nature of the VLP vaccine furthermore ensures a preferential uptake by antigen-presenting cells such as dendritic cells, and thereby an efficient presentation of DIII- as well as AP205-derived epitopes on MHC class II for the priming of specific TH cells. Activation of antigen-presenting cells is also enhanced by bacterial RNA, which is spontaneously packaged into the VLP carrier during the recombinant expression and assembly process. Upon uptake by B cells and APCs, the RNA is co-delivered with the AP205 particle to the endosomal compartment, where it can activate TLR3 or TLR7/8 (for review see [38, 39]).
In addition to its good immunogenicity, the DIII-C-AP205 vaccine is also expected to be safe and well tolerated. In contrast to live vaccines based on attenuated viruses, which inevitably carry the risk of genetic recombination and mutation into a more virulent form, DIII-C-AP205 is based on non-replicating virus-like particles derived from a bacteriophage, which are unable to infect mammalian cells. Moreover, both the VLP carrier and the antigen components of the vaccine can be produced in large amounts in bacterial expression systems and purified with relatively simple biochemical methods, suggesting that large scale production of the conjugate vaccine can be achieved in a cost-effective manner. A highly immunogenic, yet safe and affordable WNV vaccine would be attractive for veterinary prophylaxis and might also be used in elderly or immunocompromised individuals in high-risk areas. The high immunogenicity of the VLP vaccine might also offer the potential of inducing cross-protection against related flaviviruses such as Japanese encephalitis virus or dengue virus. That cross-protection may occur in principle has been shown by immunization of mice with recombinant domain III of the WNV E protein . The increased immunogenicity of the domain III by conjugation to the VLP carrier may therefore be sufficient to confer cross-protective immunity.
In the present study we show that the immunogenicity of DIII of the WNV E protein can be strongly enhanced by conjugation to virus-like particles of the bacteriophage AP205. In contrast to other vaccination approaches based on recombinant DIII, which require multiple injections and/or strong adjuvants for the induction of neutralizing antibodies, a single injection of the conjugate DIII-C-AP205 vaccine in Alum was sufficient to induce a significant amount of virus-neutralizing antibodies in mice. Three injections of the vaccine completely protected mice from a lethal WNV challenge, even when given in the absence of any adjuvant. The relatively low production costs of the DIII-C-AP205 vaccine, its superior immunogenicity with respect to other DIII-based approaches and its anticipated good safety profile make it an attractive candidate for WNV prophylaxis both in humans and in veterinary applications.
Expression and Purification of AP205 Virus-like Particles
Cleared bacterial lysates containing the recombinantly expressed coat protein of AP205 were dialysed against AEX loading buffer (20 mM NaH2PO4 pH 7.2) and loaded on a Fractogel™ TMAE column (Merck). After removal of host cell proteins by a salt wash with 333 mM NaCl, viral capsids were eluted with 600 mM NaCl and dialyzed against HAp loading buffer (5 mM NaH2PO4, 100 mM NaCl, pH 6.8). Capsids were bound to a hydroxyapatite column (Macro-prep ceramic hydroxyapatite type II, Biorad) and eluted with 60 mM NaH2PO4, 220 mM NaCl, pH 6.8, resulting in depletion of bacterial LPS.
Expression and Purification of Recombinant Domain III of the E Glycoprotein of WNV
A DNA fragment encoding domain III of the glycoprotein E of WNV NY99 was amplified from plasmid pTRHis2A-WNV-E  with the oligonucleotide pair WNV1/WNV2 (5'-ATATATCATATGGAAAAATTGCAGTTGAAGG-3'; 5'-ATATATCTCGAGTTTGCCAATGCTGCTTCCAG-3', NdeI and XhoI restriction sites are in bold) and cloned into the expression vector pET42T . The resulting plasmid encoded a fusion protein consisting of domain III of the WNV E protein (corresponding to amino acids 582-696 of the WNV polyprotein precursor), a hexahistidine tag, and a short C-terminal, cysteine containing linker (DIII-C). E. coli BL21 DE3 cells were transformed with this plasmid, and protein expression was induced in a logarithmic phase culture by addition of isopropyl-β-D-thiogalactopyranoside to a final concentration of 1 mM. After overnight growth bacteria were harvested by centrifugation, resuspended in 50 mM NaH2PO4, 150 mM NaCl, 10 mM MgCl2, 0.25% Triton X-100, pH 7.2, and lysed by sonication. Nucleic acids were digested by 1 h incubation at room temperature with 1500 U Benzonase (Sigma-Aldrich), and inclusion bodies containing recombinant DIII-C were harvested by centrifugation. After three washes with 100 mM Tris-Cl, 5 mM EDTA, 5 mM DTT, 2% Triton X-100, pH 7.0, inclusion bodies were solubilized in 8 M urea, 100 mM Tris-Cl, 100 mM DTT, pH 8.0, and loaded on a Ni-NTA column (Qiagen), which had been previously equilibrated with 8 M Urea, 100 mM NaH2PO4, 10 mM Tris-Cl, 2 mM β-mercaptoethanol, pH 8.0. Bound DIII-C was eluted with 8 M urea, 100 mM NaH2PO4, 10 mM Tris, 2 mM β-Mercaptoethanol pH 4.5, and dialysed against 2 M urea, 50 mM NaH2PO4, 0.5 M arginine, 0.5 mM oxidized glutathione, 5 mM reduced glutathione, 10% glycerol, pH 8.5. DIII-C was then refolded by stepwise dialysis against 50 mM NaH2PO4, 0.5 M arginine, 0.5 mM oxidized glutathione, 5 mM reduced glutathione, 10% glycerol, pH 8.5, and against 50 mM NaH2PO4, 10% glycerol, pH 8.5.
Chemical Cross-linking of Recombinant DIII-C to AP205 Virus-like Particles
AP205 VLPs (in PBS, pH 7.2) were first reacted for 1 h at room temperature with a 2.5 fold molar excess of the heterobifunctional cross-linker succinimidyl-6-(β-maleimidopropionamido)hexanoate (Pierce). Free cross-linker was removed by dialysis against PBS, pH 7.2. Recombinant DIII-C was incubated for 1 h at room temperature with an equimolar amount of tri(2-carboxyethyl)phosphine-hydrochloride. Under these mildly reducing conditions the cysteine residue contained in the linker is reduced, while the internal disulfide bridge of DIII-C remains intact. The reduced protein was then mixed with the derivatized AP205 VLPs at a molar ratio of 1 DIII-C monomer per 2 AP205 monomers and incubated over night at 17°C to allow cross-linking. Free DIII-C was removed by extensive dialysis against PBS pH 7.2 using cellulose ester membranes with a cut-off of 100 kDa (Spectrum Laboratories). The conjugate vaccine was analyzed by SDS-PAGE followed by Coomassie Blue staining or by Western Blot using AP205- and His-tag- specific antisera. The molecular masses of the DIII-C and AP205 monomers are similar; 13.5 kDa and 14.0 kDa, respectively. The coupling product comprising one AP205 monomer covalently conjugated to one DIII-C monomer co-migrates with the AP205 dimer band. Hence the coupling efficiency could not simply be calculated by densitometry of protein bands on a reducing SDS-PAGE stained with Coomassie Blue. Instead the conjugate vaccine was loaded on a non-reducing non-denaturing SDS-PAGE side by side with the corresponding amount of free DIII-C, which had been used in the cross-linking reaction. By comparing the intensities of the DIII-C monomers before and after cross-linking to AP205 by densitometry, the amount of DIII-C coupled to the AP205 carrier could then be quantified.
Analysis of DIII-C, AP205 and DIII-C-AP205 by Size Exclusion Chromatography
A superdex 75 column (GE Healthcare) was calibrated with a mixture of Dextran Blue (~2000 kDa), BSA (67 kDa), Ovalbumin (43 kDa), Chymotrypsinogen (25 kDa), and RNase A (14 kDa). AP205 VLPs, purified DIII-C protein and the conjugate vaccine DIII-C-AP205 were then sequentially analysed on the same column. The apparent molecular weight of DIII-C was calculated from a standard curve obtained by plotting the logarithm of the molecular weights of the protein standards against their partition coefficients.
Immunogenicity of DIII-C-AP205
Female BALB/c mice (8 weeks of age) were purchased from Charles River Laboratories. DIII-C-AP205 vaccine or the mixture of the non-conjugated vaccine components AP205 and recombinant DIII-C were diluted in PBS to 200 μl and injected subcutaneously (100 μl on two ventral sites) in the absence of additional adjuvants. Sera from immunized mice were serially diluted in PBS containing 0.05% Tween-20, 2% BSA, and applied to ELISA plates (Nunc) that had been coated with 1 μg/ml recombinant DIII-C protein. Reactivity of serum antibodies with the target protein was determined using a HRP-conjugated goat anti-mouse IgG secondary antibody (Jackson ImmunoResearch Laboratories) at a dilution of 1:1000 in PBS/0.05% Tween-20/2% BSA. After development with 1,2-phenylenediamine dihydrochloride (0.4 mg/mL in 0.066 M Na2HPO4, 0.035 M citric acid, 0.01% H2O2, pH 5.0) the optical density at 450 nm (OD450 nm) was determined using an ELISA reader (Biorad). Titers were expressed as the reciprocal of those serum dilutions that lead to half-maximal OD450 nm (OD50%).
Female C57BL/6 mice (6 weeks of age) were purchased from Harlan and allowed to acclimate to the facility for one week before experiments were performed. Experiments were approved by the animal ethics committee of the Erasmus MC Rotterdam, The Netherlands. Mice were immunized as indicated in the legend of Figure 4 and challenged two weeks after the last immunization by an intraperitoneal injection of a lethal dose of WNV-NY99 (1 × 106 TCID50). After the challenge, mice were maintained in isolation cages and observed daily for illness and death for a period of 14 days. Blood was collected on days 3 and 7 after infection and viral titers were determined by real-time PCR. The quantity of viral RNA was measured with a one-step RT-PCR TaqMan protocol and ABI PRISM 7500 detection instrument (EZ-kit, Applied Biosystems). The primers and probe used for WNV RNA quantification were: forward primer 5'-TCACTGTCAACCCTTTTGTTTC-3'; reverse primer 5'-AAGGGTGGTTCCAATTCAATC-3'; probe 5'-CCACGGCCAACGCTAAGGTCC-3'. Serial dilutions of WNV stock were used as standard, and results were expressed as TCID50 equivalents per gram of brain tissue. For determination of neutralizing antibody titers serial two-fold dilutions of immune sera were incubated with 100 TCID50 of WNV strain NY99. Virus-neutralizing titers were expressed as the reciprocal of the highest dilution that still resulted in 100% suppression of the cytopathic effects on Vero E6 cells .
Part of this work has been supported by a grant of the European Community (contract LSHB-CT-2004-005246 "COMPUVAC").
The authors would like to thank Alexander Link for critical reading of the manuscript.
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