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West Nile virus (WNV) is an emerging pathogen that is now the leading cause of mosquito-borne and epidemic encephalitis in the United States. In humans, a small percentage of infected individuals develop severe neuroinvasive disease, with the greatest relative risk being in the elderly and immunocompromised, two populations that are difficult to immunize effectively with vaccines. While inactivated and subunit-based veterinary vaccines against WNV exist, currently there is no vaccine or therapy available to prevent or treat human disease. Here, we describe the generation and preclinical efficacy of a hydrogen peroxide (H2O2)-inactivated WNV Kunjin strain (WNV-KUNV) vaccine as a candidate for further development. Both young and aged mice vaccinated with H2O2-inactivated WNV-KUNV produced robust adaptive B and T cell immune responses and were protected against stringent and lethal intracranial challenge with a heterologous virulent North American WNV strain. Our studies suggest that the H2O2-inactivated WNV-KUNV vaccine is safe and immunogenic and may be suitable for protection against WNV infection in vulnerable populations.
In the United States between 1999 and 2012, an estimated 3 million people were infected with West Nile virus (WNV), resulting in over 780,000 illnesses, greater than 36,500 confirmed cases, and 1,500 deaths (1; http://www.cdc.gov/ncidod/dvbid/westnile/index.htm). While the past few years have been characterized by lower-level endemic transmission to humans, 2012 has witnessed new and intense outbreaks of WNV neuroinvasive disease in several regions of the United States and Europe. While most WNV-infected individuals experience a mild or self-limiting febrile illness, a fraction (~1%) of cases progress to severe neurological manifestations, including high and sustained fever, headache, myalgia, meningitis, encephalitis, or acute flaccid paralysis (2). Advanced age and immunosuppression are risks for the development of severe WNV disease (2–5). Although the worldwide incidence of WNV infection is increasing (6), there is no specific treatment or vaccine available for use in humans (7).
WNV is a member of the Flaviviridae family of positive-stranded RNA viruses, which includes the globally relevant human pathogens dengue virus (DENV), yellow fever virus (YFV), and Japanese encephalitis virus (JEV). WNV was originally isolated from a febrile patient in Uganda in 1937, and phylogenetic analysis separates WNV into five distinct but genetically related lineages based on nucleic acid sequence divergence (8–11). Lineage 1 strains are considered emerging and associated with outbreaks of neuroinvasive disease (10, 12); they originate from diverse geographic regions and include WNV New York (WNV-NY), which initiated the WNV epidemic in the United States, and WNV Kunjin (WNV-KUNV), which is a nonvirulent strain of WNV circulating in Australia (10, 11, 13). Lineage 2 strains are usually less pathogenic (8), although variants, including some contemporary strains in Greece and Eastern Europe (14), periodically cause severe neurological disease (15). Less is known about lineage 3, 4, or 5 viruses, as few isolates exist, although symptomatic human cases have been attributed to them in Austria (16), Russia (17), and India (9).
Protection against primary WNV infection or secondary challenge is linked to the induction of protective humoral and cellular immune responses. Most of the protective antibodies generated against WNV bind to the structural envelope (E) protein, with a smaller subset directed against premembrane (prM) and the nonstructural proteins (reviewed in reference 18). Multiple reports have shown that WNV-specific T cells also contribute to protection and clearance of WNV from infected hosts (19–25). These studies suggest that an effective WNV vaccine should stimulate both humoral and T cell responses to achieve comprehensive protection against WNV challenge, especially in vulnerable populations. Indeed, live-attenuated replicating vaccines are immunogenic in animals and healthy adults and elicit robust adaptive immune responses (26, 27). Nonetheless, a challenge to their development and licensure is that the main target population in humans (the elderly and immunosuppressed) may be poor candidates for this class of vaccine, unless extensive safety studies are performed (28).
WNV-Innovator is a veterinary vaccine administered to horses and exotic zoo animals (29). One potential limitation to the use of this formalin-inactivated vaccine in humans is that it is generated from the highly virulent North American WNV-NY strain. A vaccine based on a virulent WNV-NY strain necessitates the generation of high-titer virus stocks for inactivation in biosafety level 3 (BSL3) facilities, which adds to production costs, and an absolute requirement for inactivation to prevent transmission of viable virulent virus into vulnerable vaccine recipients. An alternative to using virulent WNV-NY is to develop a vaccine based on a naturally attenuated strain, such as WNV-KUNV (30–32).
WNV-KUNV was first isolated in North Queensland, Australia, in 1960. Although 2.5% of the population in northern Australia is seropositive for WNV-KUNV, there has been no documented fatal human infection with this virus (33, 34), and only a very few individuals have developed WNV fever (35). WNV-KUNV is genetically similar to WNV-NY (~98% amino acid identity), with virtually complete conservation of all neutralizing antibody epitopes (11, 36). Indeed, immunization of mice with live and infectious WNV-KUNV, which itself is avirulent in immunocompetent adult mice, completely protects against lethal infection by North American WNV isolates (31). These observations suggest that WNV-KUNV may be a suitable strain for development of a vaccine in humans against the more virulent strains that circulate in the United States and other parts of the globe. Herein, we describe the preclinical evaluation of a newly developed H2O2-inactivated WNV-KUNV vaccine in adult and aged mice.
Eight- to 10-week-old C57BL/6 and BALB/c mice were obtained commercially (Jackson Laboratory). Eighteen-month-old C57BL/6 mice were purchased from the National Institute on Aging breeder colony (Harlan). HLA-A2 transgenic (HHDII) mice have been described previously (37) and were provided by T. Hansen and B. Carreno (St. Louis, MO) with permission from F. A. Lemonnier (Paris, France). These mice express a chimeric monochain of HLA-A*0201 (α1/α2 domains), mouse Db (α3 domain), and linker-attached human β2-microglobulin (β2m) and were bred in a specific-pathogen-free facility at Washington University. All mouse infections were performed in an A-BSL3-accredited facility at Washington University in accordance with federal guidelines and approval of the Washington University Animal Studies Committee.
The WNV lineage I New York isolate (WNV-NY; 3000.0259, 2000, passage 2) was described previously (38). The WNV-KUNV isolate (CH16532) was a generous gift of R. Tesh (World Reference Center of Emerging Viruses and Arboviruses, Galveston, TX). For live virus infections in mice with WNV-KUNV, 106 PFU was injected in 100 μl via the intraperitoneal (i.p.) route. For live virus infection in mice with WNV-NY, 102 PFU in 50 μl was injected subcutaneously (s.c.) via footpad injection. For intracranial (i.c.) challenges, aged mice were given 104 PFU (10,000 times the 90% lethal dose [LD90]) and young mice were administered 106 PFU (1,000,000 times the LD90) in 10 μl.
Serum-free-adapted (VP-SFM medium; Life Technologies) WHO-Vero cells (10-87; ATCC) were grown to confluence in flat-stock cell culture or on Cytodex-1 microcarrier beads (GE Healthcare) in a WAVE bioreactor (GE Healthcare). Cells were infected with WNV-KUNV strain CH16532 (multiplicity of infection, 0.1) and harvested approximately 48 h later. Supernatants were concentrated and initially purified by tangential flow filtration followed by diafiltration into inactivation buffer (20 mM Tris-HCl, pH 8.0, 50 mM NaCl, 2% sorbitol). Following sterile filtration (pore size, 0.22 μm) to remove potential aggregates, WNV-KUNV was inactivated with H2O2 (Fisher Scientific) (H2O2-WNV-KUNV) at a final concentration of 3.0% for 7 to 8 h at room temperature, with an additional filtration step performed approximately midway through the inactivation period (4 h). After inactivation, H2O2 was removed through further purification by dialysis, followed by pelleting of the virus by ultracentrifugation (200,000 × g, 16 h) or ion-exchange chromatography (cellufine sulfate; JNC Corporation). Mice were immunized i.p. with H2O2-WNV-KUNV containing approximately 1 × 107 PFU equivalents/μg protein) that was adjuvanted with either 5 μg monophosphoryl lipid A (MPL; InvivoGen) or 0.1% aluminum hydroxide gel (alum; Sigma-Aldrich). WNV-Innovator, a formalin-inactivated crude virus preparation (New York 1999 strain VM2, serial number 1666142A), is available commercially (Pfizer) and contains a proprietary oil adjuvant, MetaStim, as well as excipients. For experiments in this study with WNV-Innovator, we used a dose of 100 μl, which is 1/10 of the dose administered to horses.
To determine the kinetics of virus inactivation, WNV-KUNV that was concentrated by tangential flow filtration was treated with a final concentration of 3.0% H2O2 and monitored over time. At specific time points, aliquots were sampled from the bulk suspension and treated with catalase (final concentration, 12.5 U/ml; 10 min at room temperature; MP Biomedical) to remove residual H2O2. This procedure was performed twice on each sample to ensure complete removal of H2O2. Following this, standard plaque assays were performed on Vero cells as previously described (39). Of note, catalase treatment alone had no impact on the infectivity of WNV-KUNV (data not shown); thus, virus inactivation was due to exposure to 3.0% H2O2.
Reducing SDS-PAGE was performed with NuPAGE 4 to 12% bis-Tris gels in MOPS (morpholinepropanesulfonic acid) running buffer per the manufacturer's instructions (Life Technologies). Vaccine antigen samples were diluted into NuPAGE SDS sample buffer supplemented with a final concentration of 50 mM dithiothreitol (DTT), heated at 80°C for 10 min, and loaded onto prepared gels. Gels were run in an Xcell SureLock system (Life Technologies) for approximately 45 min at a constant voltage of 200 V and then stained with a Pierce silver stain kit (Thermo Scientific). Gel electrophoresis for Western blot analysis was carried out similarly, but DTT was omitted to maintain nonreducing conditions. Following electrophoresis, gels were blotted onto polyvinylidene difluoride membranes using an iBlot dry blotting system (Life Technologies). After transfer, membranes were blocked (5% nonfat dry milk in phosphate-buffered saline [PBS], 0.05% Tween 20 [PBS-T]) for 1 h at room temperature and probed with the WNV-specific monoclonal antibody (MAb) 7G11 at 1 μg/ml in blocking buffer for 1 h at room temperature. Membranes were washed five times with PBS-T and probed with a horseradish peroxidase-conjugated goat anti-mouse IgG secondary antibody at an optimal dilution for 1 h at room temperature. Following a final series of washes with PBS-T, blots were developed with the Pierce ECL Western blotting substrate (Thermo Scientific) according to the manufacturer's recommendations and exposed to Biomax XAR film (Kodak).
CD8+ T cells were depleted from mice with a rat MAb (53-5.8; rat IgG2b; BD Bioscience) specific for the mouse CD8β chain according to a previously published protocol (40). A rat IgG1 antibody (Jackson ImmunoResearch) was used as an isotype control. Anti-CD8β or isotype control MAb (40 μg) was administered to mice via an i.p. injection 2 days prior to i.c. challenge, and an additional 10 μg of anti-CD8β or isotype control MAb was administered intravenously (i.v.) on the day of virus challenge. Two days after infection, the efficiency of depletion of CD8+ T cells was assessed by flow cytometry after staining peripheral blood mononuclear cells with anti-CD8α (Biolegend).
Intracellular staining for tumor necrosis factor alpha (TNF-α) and gamma interferon (IFN-γ) of lymphocytes isolated from the spleen was performed as described previously (41). Briefly, spleens were harvested and homogenized to form a single-cell suspension and incubated with 2 μg/ml brefeldin A (Sigma) for 6 h at 37°C with 10−6 M of peptide NS4B from residues 2488 to 2496, E from residues 347 to 354, E from residues 771 to 778 (20) (H-2b mouse CD8+ T cells), or SVG9 E from residues 720 to 728 (42) (HLA-A2 transgenic CD8+ T cells) or 2 μg/ml anti-CD3 (145-2C11) (BD Biosciences). After incubation, the cells were stained with directly labeled antibodies (all from Biolegend, unless indicated) against CD3, CD4, CD11a, CD19, CD27, CD43, CD62L, CD69, CD122, CD127, CD8α, CD44, programmed cell death protein 1 (PD-1), killer cell lectin-like receptor subfamily G member 1 (KLRG1), and cytotoxic T lymphocyte antigen 4 (CTLA-4). Db-NS4B tetramer and HLA-A2 SVG9 tetramer were obtained from the NIH tetramer core facility. Cells were washed, fixed, permeabilized with FixPerm buffer (eBioscience), and stained intracellularly for anti-IFN-γ, anti-TNF-α, (eBioscience), or anti-granzyme B (Invitrogen). Lymphocytes were processed on an LSRII flow cytometer (BD Bioscience) using FACSDiva (version 6.1.1) software (BD Bioscience) and analyzed with FlowJo software (Treestar). The total numbers of IFN-γ- or TNF-α-expressing CD8+ T cells was determined by multiplying the percentage of IFN-γ-positive (IFN-γ+) or TNF-α-positive (TNF-α+) CD8+ T cells by the total numbers of splenocytes.
WNV-specific IgG levels were determined using an E-protein-specific enzyme-linked immunosorbent assay (ELISA) as described previously (43). Briefly, individual wells of a microtiter plate were coated overnight at 4°C with 1 μg/ml of recombinant WNV E protein. Plates were washed and blocked with 1% bovine serum albumin (BSA) in PBS supplemented with 0.05% Tween 20 (blocking buffer). Serum samples from naïve, immunized, or infected mice were heat inactivated (56°C for 60 min), serially diluted in blocking buffer, and added to wells for 1 h at room temperature. Following several washes, biotin-conjugated goat anti-mouse IgG (Sigma-Aldrich) was added for 1 h at room temperature. Plates were then washed and incubated with streptavidin-conjugated horseradish peroxidase (2 μg/ml in blocking buffer; Vector Laboratories) for 1 h at room temperature. After several washes, plates were developed with tetramethylbenzidine substrate (Dako) and the reaction was stopped with 1 N H2SO4. The optical density at 450 nm was measured, and endpoint dilutions were defined as those that were twice above the background average (BSA-coated wells).
The neutralizing activity of serum antibodies against WNV was assessed using either a focus or a plaque reduction neutralization assay (44). Neutralization titers of serum from BALB/c mice were determined using a plaque reduction assay as described previously (45). For studies in aged C57BL/6 mice, 50 focus-forming units of WNV were preincubated with serial dilutions of heat-inactivated mouse serum at 37°C for 1 h in Dulbecco modified Eagle medium with 2% fetal bovine serum (FBS), penicillin, and streptomycin. Virus-serum mixtures were added in triplicate to individual wells of 96-well tissue culture plates containing Vero cell monolayers at ~90% confluence. Virus was incubated with Vero cells for 1 h at 37°C, after which wells were overlaid with 1% carboxymethyl cellulose (Sigma) in minimal essential medium supplemented with 4% FBS. After culture at 37°C for 24 h, cells were fixed with 1% paraformaldehyde in PBS (10 min at room temperature) and permeabilized with 0.1% saponin and 0.1% BSA in PBS (saponin buffer; 5 min at room temperature). Cells were stained with the humanized E16 anti-WNV MAb (46) (50 μl at 200 ng/ml in saponin buffer) for 2 h at room temperature. Following several washes, wells were incubated with horseradish peroxidase-conjugated anti-human IgG antibody (1:5,000 in saponin buffer; Sigma) for 1 h at room temperature. Wells were washed and infectious foci were visualized with TrueBlue substrate (KPL) after a 5- to 10-min incubation at room temperature. Wells were rinsed with water and dried prior to analysis with a Biospot counter (Cellular Technology) using Immunocapture software. The percent reduction in spot numbers in samples preincubated with serum compared to the numbers in wells with virus preincubation with medium alone were graphed using Prism software, and 50% effective concentration (EC50) values were calculated.
For survival analysis, Kaplan-Meier curves were analyzed by the log rank test. The statistical significance of viral burden, antibody titers, and number of activated T cells was analyzed by the Mann-Whitney test. All statistical analyses were performed using Prism software (GraphPad Prism).
Our goal was to generate an inactivated immunogenic vaccine that elicited protective immune responses against WNV and could be used safely in at-risk populations. Previously, members of our group demonstrated that an H2O2-inactivated WNV-NY could induce effective neutralizing antibody responses in BALB/c mice (39). Although inactivated WNV-NY might be a suitable veterinary vaccine, for humans, given the possible safety concerns and manufacturing issues associated with large-scale cultivation of a BSL3 virus, we decided to use the attenuated WNV-KUNV strain, as it has 98% amino acid identity to WNV-NY (10, 36). To inactivate WNV-KUNV, a previously determined optimal concentration of H2O2 (3.0%, vol/vol) (39) was added to concentrated virus for a period of 7 to 8 h. Kinetic inactivation experiments demonstrated that under these conditions, WNV-KUNV showed a rapid loss of infectivity, with a half-life of approximately 2.6 min (Fig. 1A). Based on these inactivation kinetics, we estimated that 7 h of inactivation would achieve a greater than 10-log-unit reduction of infectious virus. However, since the amount of viable virus dropped below our limit of detection within 90 min of the inactivation procedure (Fig. 1A), additional coculture assays in Vero cells, using up to 5% of the final purified vaccine, confirmed complete inactivation (data not shown). Following inactivation, the vaccine material was purified further by ultracentrifugation and ion-exchange chromatography. Analysis by SDS-PAGE of the final purified, inactivated vaccine demonstrated that it contained three bands at approximately 55, 20, and 15 kDa, which correspond to the predicted molecular masses of the envelope (E), premembrane, and capsid (C) proteins, respectively (Fig. 1B). The identity of the dominant 55-kDa band was confirmed by Western blotting with an anti-WNV E-protein-specific MAb (Fig. 1C). Representative vaccine lots were screened for recognition by conformationally sensitive MAbs against WNV and indicated that the H2O2-inactivated virus remains antigenically intact (data not shown).
To assess the potential of the H2O2-WNV-KUNV vaccine to generate a neutralizing antibody response, 8-week-old BALB/c mice (n = 5 for each dose) were immunized with a single dose of 2.5, 10, or 40 μg of vaccine complexed with 0.1% alum. Neutralization assays were performed with serum harvested from animals at days 28 and 90 after vaccination (Fig. 2A). By day 28, H2O2-WNV-KUNV induced neutralizing antibody responses against WNV-KUNV in a dose-dependent manner (mean ± standard error of the mean [SEM] 50% neutralization titers [NT50], 23,068 ± 6,865 for a dose of 40 μg, 2,586 ± 1,068 for 10 μg, and 251 ± 27 for 2.5 μg). At 90 days postimmunization, neutralization titers had increased at all three doses tested (mean ± SEM NT50 values, 103,602 ± 23,825 for a dose of 40 μg, 6,958 ± 2,042 for 10 μg, and 4,506 ± 1,608 for 2.5 μg). Thus, immunization with H2O2-WNV-KUNV, analogous to H2O2-WNV-NY (39), induced high titers of neutralizing antibodies against WNV in mice that were detected at least 3 months after single-dose vaccination.
The majority of neutralizing antibodies against WNV are directed against the E protein (12, 46–48) and inhibit infection by blocking virus attachment, entry, and fusion (reviewed in references 49 and 50). To further define the humoral response after vaccination with H2O2-WNV-KUNV (10 μg) formulated with alum, we assessed the kinetics of induction of anti-WNV-E-specific antibodies in 8-week-old BALB/c and C57BL/6 mice with or without boosting. In this series, serum was collected on days 0, 14, 28, 42, 60, and 90 days postimmunization, and on day 28, half of the mice in each group were boosted with alum-adjuvanted H2O2-WNV-KUNV. Antibody induction was monitored using an ELISA that detected antibodies against WNV-NY E protein (Fig. 2B). Both C57BL/6 and BALB/c mice generated robust anti-WNV E-specific antibody responses by day 14 following a single administration of the vaccine (for C57BL/6 mice, 833 ± 146 reciprocal dilution of serum; for BALB/c mice, 198 ± 43 reciprocal dilution of serum), and substantially increased titers were observed on days 42, 60, and 90 in the mice that were boosted (Table 1). Higher levels (2- to 7-fold, P < 0.05) of WNV-E-specific antibodies were observed on days 14, 42, and 90 in the C57BL/6 mice than BALB/c mice that received a single dose of vaccine. However, no differences in the WNV E-specific antibodies were observed in the two strains of mice after boosting (P > 0.4).
Vaccination with H2O2-WNV-NY or H2O2-WNV-KUNV provides 100% protection against lethal WNV infection following a peripheral route of infection (39) (data not shown). To determine the protective activity of the H2O2-WNV-KUNV vaccine in a highly stringent i.c. challenge model, immunized BALB/c or C57BL/6 mice were infected i.c. with 106 PFU of WNV-NY (1,000,000 times the LD90) on day 90 after initial vaccination (Fig. 2C). As expected, all unimmunized BALB/c and C57BL/6 mice succumbed to i.c. infection. While only one (10%) BALB/c mouse that received a single dose of the H2O2-WNV-KUNV vaccine survived the challenge, 60% of C57BL/6 mice were protected (P = 0.05). In comparison, BALB/c (10 of 10) and C57BL/6 (9 of 10) mice that had been boosted on day 28 were protected against the i.c. challenge on day 90. Thus, the H2O2-WNV-KUNV vaccine induces sufficient immunity to protect mice from a highly lethal direct infection of WNV in the brain, although boosting was required for the greatest protection.
We next determined whether CD8+ T cells contributed to the protection conferred by the H2O2-WNV-KUNV vaccine. We speculated that this was possible with a nonreplicating vaccine, as a substantial fraction of the anti-WNV CD8+ T cell response associated with live virus infection requires CD8-α dendritic cells and antigen cross-presentation (41, 51). In C57BL/6 mice, the immunodominant CD8+ T cell epitope is in the NS4B protein (20, 52). As the H2O2-WNV-KUNV vaccine is highly purified, the nonstructural NS4B protein is absent from the vaccine preparation. Nonetheless, there are two subdominant epitopes (E347 and E771) that are encoded by the WNV E protein and conserved in WNV-KUNV that could potentially elicit a CD8+ T cell response (20, 52). To assess this, we measured the generation of the WNV-specific CD8+ T cell response against the NS4B, E347, and E771 peptides in C57BL/6 mice following immunization with H2O2-WNV-KUNV or infection with live WNV-NY or WNV-KUNV (Fig. 3A and andB).B). While we detected a CD8+ T cell response to the dominant and subdominant epitopes following infection with the live viruses, we did not identify a CD8+ T cell response to any of the epitopes following vaccination with purified H2O2-inactivated WNV.
We speculated that a CD8+ T cell response still might have been generated by H2O2-WNV-KUNV but was below the threshold level of detection by our flow cytometry assay. To test this hypothesis, we evaluated the effect of CD8+ T cell depletion on secondary challenge following vaccination (Fig. 3C). Groups of 16 mice were immunized with 10 μg of H2O2-WNV-KUNV, infected with 102 PFU of live WNV-NY or 106 PFU of live WNV-KUNV, or left unvaccinated. Twenty-eight days later, all mice received 40 μg of a CD8β-depleting or isotype control MAb. Two days later, the mice were administered a second dose of depleting or isotype control MAb and concurrently challenged with 106 PFU of WNV-NY via a stringent i.c. route. As expected, all naïve control mice succumbed to i.c. infection regardless of the presence or absence of CD8+ T cells. In comparison, mice receiving live WNV (WNV-NY or WNV-KUNV) survived the challenge independently of CD8+ T cell depletion. However, we observed a significant survival decrease (58 versus 14% survival, P = 0.006) in vaccinated mice receiving the CD8β-depleting MAb compared to that in mice receiving the isotype control MAb. Thus, although WNV-specific CD8+ T cells in vaccinated animals were below our limits of detection by flow cytometry, a response was generated and protected mice against a stringent lethal virus challenge.
To confirm the potential of H2O2-WNV-KUNV for generating a CD8+ T cell response and begin to address its applicability for humans, we repeated immunization studies in HHDII mice (37, 53). These mice are on an isogenic C57BL/6 background and express the α1 and α2 domains of HLA-A*0201 linked to the α3 domain of mouse Db with a linker-attached human β2m. These mice are genetically deficient in mouse β2m and H2-Db and have very low surface expression of H2-Kb. Due to the absence of mouse β2m and mouse neonatal FcR (FcRn), HHDII mice have much lower levels of circulating antibody and are highly susceptible to WNV infection, making them less useful as a pathogenesis or vaccine model in which humoral responses are important. However, they are a suitable model for assessing CD8+ T cell responses against epitopes that are restricted by HLA-A*0201 and have previously been used to demonstrate CD8+ T cell responses against WNV (53). In WNV infection of humans, the immunodominant HLA-A2-restricted epitope falls within the E protein (SVG9 [SVGGVFTSV]) (42, 53, 54) and is conserved between members of the Flaviviridae family, including WNV-NY and WNV-KUNV (data not shown).
To test for the development of HLA-A2-restricted WNV-specific CD8+ T cell responses, HHDII mice either were immunized with 40 μg of H2O2-WNV-KUNV or infected with 106 PFU of WNV-KUNV. At day 10 postinfection or postvaccination, the spleens of the HHDII mice were harvested and the CD8+ T cells were characterized functionally and phenotypically (Fig. 4A to toD).D). Using an SVG9-specific major histocompatibility complex (MHC) class I tetramer, we identified a WNV-specific CD8+ T cell population in both vaccinated and infected animals, although the percentage and total number of SVG9-specific CD8+ T cells was lower in vaccinated mice (5% compared to 11%, P = 0.04; 3.6 × 104 compared to 8.7 × 104 cells, P = 0.002; Fig. 4A). SVG9-specific CD8+ T cells from vaccinated mice were also distinct phenotypically from those generated following infection, as they expressed significantly higher levels of interleukin-7Rα (CD127, P = 0.008); PD-1 (P = 0.03), a negative regulator of immune responses; and CD62L (P = 0.04), a cell adhesion molecule that regulates T cell homing (Fig. 4B).
We next characterized functionally the antigen-specific CD8+ T cells after vaccination or infection. At day 8 postinfection or postvaccination, splenic CD8+ T cells were restimulated with SVG9 peptide and analyzed for intracellular levels of the cytokines IFN-γ and TNF-α. While CD8+ T cells from both H2O2-WNV-KUNV-vaccinated and WNV-KUNV-infected mice produced IFN-γ and TNF-α, overall, there were a higher percentage and a higher number of SVG9-specific CD8+ T cells present after live virus infection (Fig. 4C, left four panels). Nonetheless, we observed no difference in the relative proportion of antigen-specific CD8+ T cells that produced IFN-γ, TNF-α, or IFN-γ and TNF-α (double-positive cells) when comparing samples from vaccinated and infected mice, which suggests that the polyfunctionality of the CD8+ T cell response was equivalent (Fig. 4C, far right panel). To assess their relative avidity, antigen-specific CD8+ T cells from H2O2-WNV-KUNV-vaccinated or WNV-KUNV-infected mice were stimulated ex vivo with a dose titration of the SVG9 peptide, and the production of IFN-γ and TNF-α was analyzed (Fig. 4D). No difference in the functional avidity of the SVG9-specific CD8+ T cells was observed after vaccination and infection.
An ideal WNV vaccine would elicit a durable and protective immune response in the elderly and immunocompromised. However, studies with vaccines against other viruses (e.g., influenza virus) have revealed that with aging, the ability to induce protective adaptive immunity wanes (reviewed in reference 55). To begin to determine the utility of H2O2-WNV-KUNV for this target population, we vaccinated three groups of aged (age, >18 months) mice with 10 μg of H2O2-WNV-KUNV vaccine complexed with alum, 100 μl (i.e., 1/10 the equine dose) of WNV-Innovator, or alum alone. Mice were bled on days 0, 14, 28, 42, 60, and 90 postvaccination, and the serum IgG response against WNV-NY E protein was measured by ELISA (Fig. 5A). On day 28, the groups were boosted with 10 μg of H2O2-WNV-KUNV, 100 μl of WNV-Innovator, or alum, respectively. Notably, we observed no difference (P > 0.8) in the anti-E-protein antibody response between the H2O2-WNV-KUNV and WNV-Innovator vaccines at any of the time points, whereas the responses to both vaccines were significantly higher than the response to alum alone. Thus, H2O2-WNV-KUNV and WNV-Innovator both induced a robust E-protein-specific humoral response in aged C57BL/6 mice.
To assess the functional quality of the antibody response in immunized aged mice, we assessed neutralizing activity (Fig. 5B). By day 90, both vaccines had induced strongly neutralizing responses, although WNV-Innovator had a higher NT50 value than H2O2-WNV-KUNV (7,850 versus 818, P < 0.002). To determine if the immune responses generated in the aged mice were protective against i.c. challenge, these animals were infected on day 90 (age, >21 months) with 104 PFU (10,000 times the LD90) of WNV-NY (Fig. 5C). All aged mice that had received alum alone succumbed to the WNV-NY challenge, with a mean survival time of 8 days. Aged mice that received WNV-Innovator were protected (P < 0.0001). In comparison, mice that received H2O2-WNV-KUNV also showed significant protection (53% survival, P = 0.005 compared to alum alone) from i.c. challenge. Thus, both inactivated vaccines induced immunity in aged animals, and the WNV-Innovator vaccine provided greater protection in the i.c. challenge model.
The development of a WNV-specific humoral immune response is an important criterion for the development of an effective vaccine. Passive transfer of serum containing WNV-specific antibodies protects against virus dissemination into the central nervous system and prevents WNV encephalitis and death (56, 57). In humans and mice, a large component of the WNV-specific protective antibody response is directed against the viral E protein (18, 58, 59). Here, we demonstrate the protective capacity of a novel H2O2-WNV-KUNV vaccine against lethal WNV challenge. Following vaccination with H2O2-WNV-KUNV in both young and old mice, we observed a significant WNV E-protein-specific and neutralizing antibody response that was enhanced by boosting and remained elevated for the duration of the study. Moreover, using human HLA-A2 transgenic and wild-type C57BL/6 and BALB/c adult mice, we showed that the H2O2-WNV-KUNV vaccine generates a polyfunctional antigen-specific CD8+ T cell response. Taken together, H2O2-WNV-KUNV induces adaptive immunity to WNV that protects adult and aged mice against lethal infection.
We compared the antibody response of H2O2-WNV-KUNV to that of a commercially available veterinary vaccine (WNV-Innovator), which is used in horses and exotic birds but not in humans. The inactivated WNV-Innovator formulation is produced after formalin inactivation of WNV-NY and complexing with MetaStim, a proprietary adjuvant (23, 29). The vaccine, however, is not purified in a virion-only form, as in C57BL/6 mice it induced a CD8+ T cell response against the Db-restricted NS4B peptide, which is absent from the virion (23). Although WNV-Innovator is used effectively in horses (60), there are regulatory barriers to using unpurified vaccines in humans. Moreover, as the viral backbone is derived from the virulent WNV-NY 1999 strain, this raises the safety threshold for complete virus inactivation before immunization of humans, especially those at risk for severe disease (61). While a single administration of purified H2O2-WNV-KUNV induced a strong WNV-specific antibody response in C57BL/6 mice, the antibody titer was lower than that observed with WNV-Innovator. This finding differs from that of a previous study using sucrose gradient-purified H2O2-inactivated WNV-NY in BALB/c mice (39). These differences could be due to mouse strain variation (BALB/c versus C57BL/6), the virus strain used for vaccine production (WNV-NY versus WNV-KUNV), or the relative purity of the vaccine preparation. Following boosting, however, comparable WNV-specific E antibody titers were observed with H2O2-WNV-KUNV and WNV-Innovator. Regardless, the H2O2-WNV-KUNV vaccine provided significant protection in young and aged mice from a stringent i.c. challenge with WNV-NY.
CD8+ T cells contribute to protection against and clearance of primary WNV infection (20, 22, 23, 52, 62) and secondary challenge (23). We initially set out to assess whether H2O2-WNV-KUNV could stimulate both a protective antibody and CD8+ T cell response. Due to the purity of the vaccine preparation, which contains virions composed of prM/M, E, and C proteins and the viral RNA, the H-2b immunodominant epitope in the NS4B protein was absent, making it difficult to detect an antigen-specific CD8+ T cell response in C57BL/6 mice. Nonetheless, depletion studies followed by i.c. challenge revealed that the H2O2-WNV-KUNV vaccine induced a CD8+ T cell response in C57BL/6 mice. While the vaccine response generated a robust neutralizing antibody response, it was not sufficient to prevent lethal infection after direct introduction of WNV into the brain; protection required the presence of CD8+ T cells. In comparison, and as reported previously (23), mice that received live WNV or WNV-KUNV and survived initial infection produced higher levels of antibody and survived rechallenge regardless of the presence of CD8+ T cells.
H2O2-WNV-KUNV also induced a robust polyfunctional CD8+ T cell response in the HLA-A2 human MHC class I transgenic mice, wherein the immunodominant CD8+ T cell epitope against WNV maps to the SVG9 peptide in the E protein (42) and, thus, is present in the purified vaccine. We observed no difference in the relative avidity of SVG9-specific CD8+ T cells from mice receiving live WNV-KUNV in comparison to those from mice vaccinated with H2O2-WNV-KUNV. Priming of a potent cellular immune response against a human HLA-restricted WNV epitope suggests that H2O2-WNV-KUNV might stimulate a protective CD8+ T cell response in humans. In support of this, profiling studies from WNV-infected patients have shown that multiple CD8+ T cell epitopes map to the structural proteins of WNV and are presented by different MHC class I alleles (42, 54, 63, 64). Future immunization studies in other HLA transgenic mice and, ultimately, humans will be required to define the extent of antigen-specific CD8+ T cell responses generated after H2O2-WNV-KUNV administration.
The elderly and immunocompromised are at the greatest risk of developing severe neurological sequelae as a consequence of WNV infection (65). However, age-related immune defects in both the innate and adaptive immune response create hurdles to the development of an effective vaccine (see reference 66 and references therein). Similar to humans, aged mice are more susceptible to severe WNV disease and, thus, a reasonable surrogate for determining vaccine efficacy (19). Our observation that aged mice developed strong WNV-specific humoral responses and were protected after immunization and boosting with H2O2-WNV-KUNV is a first step in demonstrating vaccine efficacy in a highly susceptible population. Further studies are needed to assess whether the mechanism of protection in aged mice is similar in its relative composition (antibody versus T cell mediated) to that in younger adult mice and whether each component is durable.
Although a significant fraction of aged mice were protected 2 months after boosting with a 10-μg dose of H2O2-WNV-KUNV formulated with alum, complete protection was generated only by the formalin-inactivated WNV-Innovator vaccine formulated with a strong veterinary adjuvant, MetaStim. It is unclear how much WNV protein (soluble and intact virion protein) is in the WNV-Innovator vaccine. It is possible that the response to a higher, 40-μg dose of highly purified H2O2-WNV-KUNV could meet or exceed the neutralizing antibody responses elicited by immunization with a 1/10 horse dose of WNV-Innovator vaccine, and this could potentially improve protection against robust i.c. challenge. Alternatively, since the WNV-Innovator vaccine is not purified, it may contain other uncharacterized factors, such as the immunogenic NS4B protein (a nonstructural protein not found in purified H2O2-WNV-KUNV), that could enhance the efficacy and/or immunogenicity of the vaccine; indeed, robust NS4B-specific CD8+ T cell responses were previously observed in C57BL/6 mice immunized with the WNV-Innovator vaccine (23). At present, it is unclear if the protective advantage of the WNV-Innovator vaccine observed in aged C57BL/6 mice is due to CD8+ T cell responses against WNV-specific nonstructural proteins (e.g., NS4B) or, possibly, antibodies against other nonstructural proteins. For example, passive transfer of anti-NS1 MAbs protects against WNV infection in mice (67), and vaccination of rhesus macaques with NS1 protein from yellow fever virus provided strong protective immunity against lethal virus challenge (68). To improve the immunogenicity of H2O2-WNV-KUNV, in future studies, we plan to optimize the dose, prime-boost sequence, adjuvant, and route of administration to create an inactivated vaccine that confers the greatest level of protection in susceptible populations. Although in recent years it appeared as if WNV infection and disease might wane, the recent epidemic outbreaks in the United States and Europe in 2012 (1, 6, 14, 15, 69) suggest that this trend may be changing, highlighting a need for the development of a safe and effective WNV vaccine that can be used in at-risk populations.
NIH grants and contracts 5U01AI082196, R01AI098723, and HHSN272201100017C supported this work.
The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
While the authors M.S.D, A.K.P, and J.M.R. do not have a conflict of interest to report, M.K.S., I.J.A., P.P.P., and E.A.P. have a financial interest in Najít Technologies, Inc., a company that may have a commercial interest in the results of this research and technology. This potential individual and institutional conflict of interest has been reviewed and managed by OHSU.
Published ahead of print 5 December 2012