Vaccine 32 (2014) 800–808

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Matrix-MTM adjuvanted envelope protein vaccine protects against lethal lineage 1 and 2 West Nile virus infection in mice Sofia E. Magnusson a,∗ , Karin H. Karlsson a , Jenny M. Reimer a , Silke Corbach-Söhle b , Sameera Patel b , Justin M. Richner c , Norbert Nowotny d,e , Luisa Barzon f , Karin Lövgren Bengtsson a , Sebastian Ulbert g , Michael S. Diamond c , Linda Stertman a a

Novavax AB, Uppsala, Sweden University of Zürich, Institute of Virology, Switzerland c Departments of Medicine, Molecular Microbiology, Pathology & Immunology, Washington University School of Medicine, St Louis, USA d Institute of Virology, University of Veterinary Medicine, Vienna, Austria e Department of Microbiology and Immunology, College of Medicine and Health Sciences, Sultan Qaboos University, Muscat, Oman f Department of Molecular Medicine, University of Padova, Italy g Fraunhofer Institute for Cell Therapy and Immunology, Leipzig, Germany b

a r t i c l e

i n f o

Article history: Received 23 September 2013 Received in revised form 5 December 2013 Accepted 12 December 2013 Available online 28 December 2013 Keywords: Matrix MTM Adjuvant West Nile virus Vaccine

a b s t r a c t West Nile virus (WNV) is a mosquito-transmitted flavivirus and an emerging pathogen in many parts of the world. In the elderly and immunosuppressed, infection can progress rapidly to debilitating and sometimes fatal neuroinvasive disease. Currently, no WNV vaccine is approved for use in humans. As there have been several recent outbreaks in the United States and Europe, there is an increasing need for a human WNV vaccine. In this study, we formulated the ectodomain of a recombinant WNV envelope (E) protein with the particulate saponin-based adjuvant Matrix-MTM and studied the antigen-specific immune responses in mice. Animals immunized with Matrix-MTM formulated E protein developed higher serum IgG1 and IgG2a and neutralizing antibody titers at antigen doses ranging from 0.5 to 10 ␮g compared to those immunized with 3 or 10 ␮g of E alone, E adjuvanted with 1% Alum, or with the inactivated virion veterinary vaccine, Duvaxyn® WNV. This phenotype was accompanied by strong cellular recall responses as splenocytes from mice immunized with Matrix-MTM formulated vaccine produced high levels of Th1 and Th2 cytokines. Addition of Matrix-MTM prolonged the duration of the immune response, as elevated humoral and cellular responses were maintained for more than 200 days. Importantly, mice vaccinated with Matrix-MTM formulated E protein were protected from lethal challenge with both lineage 1 and 2 WNV strains. In summary, Matrix-MTM adjuvanted E protein elicited potent and durable immune responses that prevented lethal WNV infection, and thus is a promising vaccine candidate for humans. © 2013 Elsevier Ltd. All rights reserved.

1. Introduction West Nile virus (WNV) is a positive-stranded, enveloped RNA flavivirus and an emerging pathogen [1,2]. WNV cycles in nature between birds and mosquitoes but causes disease in vertebrate animal species, including humans and horses [1]. WNV has now spread globally with outbreaks in central and southeast Europe, Asia, Africa and North America [3–6]. In a small fraction of WNV infected individuals, infection can progress to severe neurological disease and death, especially in elderly and immunosuppressed individuals [7].

∗ Corresponding author at: Novavax AB, Kungsgatan 109, SE-75318, Uppsala, Sweden. Tel.: +46 18 16 17 46; fax: +46 18 16 17 01. E-mail address: [email protected] (S.E. Magnusson). 0264-410X/$ – see front matter © 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.vaccine.2013.12.030

Although there is currently no WNV vaccine approved for human use, veterinary vaccines already exist and preclinical and early-phase clinical trials in humans have been performed with recombinant protein, DNA plasmid, and live attenuated strain vaccine candidates [8–20]. Given the continuing outbreaks of severe WNV disease in the United States and southeastern Europe, there is a need for a safe and effective human WNV vaccine, especially one that targets at-risk elderly and immunocompromised populations. Here, we formulated the ectodomain of a recombinant WNV envelope (E) protein from the lineage 1 WNV strain New York 1999 with the adjuvant Matrix-MTM and assessed its ability to induce protective immune responses in mice. Matrix-MTM is a novel adjuvant formulated with saponin, cholesterol and phospholipids into a 40 nm cage-like structure. Saponins obtained from the bark of the tree Quillaja saponaria Molina have immune stimulatory properties and Quillaja saponins

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have been used successfully for decades in animal vaccines [21,22]. Particulate saponin adjuvants such as Matrix-MTM augment Th1 and Th2 responses, activate immune cells, enhance immune cell trafficking and have the typical adjuvant effect of antigen dosesparing [23–27]. Matrix-MTM has been evaluated in two Phase I human studies on influenza vaccines. Both studies showed promising results with increased humoral and cellular responses along with excellent safety data ([28] and Lövgren et al. unpublished results). We investigated the immune response and efficacy of MatrixMTM adjuvanted WNV E protein in mice using different vaccine antigen doses and immunization schemes. Adjuvanting WNV E protein with Matrix-MTM enhanced total and neutralizing antibody titers, skewed antibody isotypes toward those with enhanced effector function capacity, augmented cellular recall responses, and protected mice in two separate lethal challenge models using WNV lineage 1 and 2 strains, respectively. 2. Materials and methods 2.1. Adjuvants and vaccine antigen Matrix-MTM (Novavax AB (formerly Isconova AB), Uppsala, Sweden) is composed of two 40 nm large particles made from two separate saponin fractions, i.e. Matrix-ATM and Matrix-CTM . Alum (Al(OH)3 ) was purchased from a commercial source (Alhydrogel 2%, Brenntag, Fredrikssund, Denmark). The WNV E ectodomain (amino acid residues 1 to 404) of the New York 1999 strain was cloned into the pET21a bacterial expression plasmid, expressed in Escherichia coli, and purified after an oxidative refolding protocol, as described previously [29]. E protein was isolated as a monodispersed peak on a Superdex 75 or 200, 16/60 size-exclusion column using fast-protein liquid chromatography (GE Healthcare). Prior to vaccination the E protein was tested for endotoxin levels using a Limulus Amebocyte Lysate assay (Endosafe® KTA2TM , USA). The endotoxin units (EU) ranged from 0.024 EU/␮g to 0.078 EU/␮g in the different E batches. Matrix-MTM was mixed with E at the day of injection and Alum-adjuvanted E was prepared the day before injection.

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mice in separate cages and each group was repeated three independent times (total of n = 9–10 per group). Blood samples for serology were taken at day 21 or 38 after primary vaccination. Mice were inspected daily for clinical signs of WNV infection and euthanized 20 days after infection or earlier depending on whether the end point score was reached. 2.3. ELISA Quantification of WNV E-specific IgG1 and IgG2a antibodies was performed by ELISA, as described previously [24] using Maxisorp plates (Nunc) coated with 0.1 ␮g of E protein/well. IgG1 and IgG2a anti-E titers were calculated using a four-parameter logistic equation (Softmax software, Molecular Devices). The inflection point of the titration curve was taken as titer value (logarithmic reciprocal titer). The lowest dilution, 1:30, i.e. 1.477 when logarithmic, was set as limit of detection for the assay. 2.4. Neutralizing antibody assays The serum neutralizing antibody titers were determined by plaque reduction neutralization test (PRNT) or microtiter virus neutralization (VN) assay, as described in the World Organization for Animal Health (OIE) Manual [31]. The PRNT was performed in Vero cells cultured in 6-well plates essentially as described [32]. Serum samples were tested in duplicate at two-fold dilutions starting from 1/20 and mixed with an equal volume of 50 PFU/well of the WNV reference strain Eg101. Determination of PRNT50 and PRNT90 endpoint titers was based on the serum dilution that led to 50% and 90% reduction, respectively, of the number of plaques compared with the virus control wells, which exhibited approximately 50 plaques. The microtiter VN assay was performed in Vero cells cultured in 96-well microtiter plates. Serum samples were tested in triplicate at two-fold dilutions starting from 1/20 and mixed with an equal volume of 100 infectious units (measured as 50% tissue culture infective doses, TCID50) of WNV Eg101. After incubation for 5 days, cells were stained with crystal violet and the plates read using an inverted microscope. Wells were scored for the degree of cytopathic effect (CPE) observed. The neutralization titer (NT) was determined as the highest serum dilution able to neutralize more than 90% CPE.

2.2. Mice, immunizations and challenge 2.5. Ex vivo re-stimulation of splenocytes and cytokine analysis Female BALB/c mice (8–12 week-old) were purchased from Scanbur (Sweden) and housed at the National Veterinary Institute (SVA, Sweden) or purchased from Charles River (Germany) and housed at the Institute of Laboratory Animal Science (University of Zürich, Switzerland). All animal experiments were approved by the local Ethics committees and animals were maintained in accordance with national guidelines. Mice were immunized subcutaneously (s.c.) at the base of the tail in groups of 6–12 mice with either 0.5, 1, 3 or 10 ␮g of WNV E protein adjuvanted with or without 5 or 10 ␮g of Matrix-MTM . In one experimental series, 8 mice per group received 3 ␮g of E protein adjuvanted with 1% Alum or 1/10th of a horse dose of the veterinary vaccine Duvaxyn® WNV (current name Equip WNV, Pfizer), a formalin-inactivated whole virus vaccine, s.c. at the base of the tail as a comparison. Booster immunizations were given s.c. at day 28 after the first immunization when indicated. In challenge studies, 8 week-old female BALB/c mice were injected intraperitonealy (i.p.) with 200 ␮l of 104 plaque forming units (PFU) of WNV lineage 1 strain ITA-09 [30] or 200 ␮l of 105 PFU of WNV lineage 2 strain AUT-08 [3] 42 days after primary vaccination and 14 days after boosting with 3 or 10 ␮g E adjuvanted with 10 ␮g Matrix-MTM or with 1/10th of a horse dose of Duvaxyn® WNV, used as a comparison vaccine. Control mice were injected with adjuvant only. Animals were housed in groups of three or four

Splenocyte suspensions were prepared as described [24] and re-stimulated with E protein in vitro. Briefly, 0.5 × 106 splenocytes/well in 96-well plates were cultivated in RPMI medium with 5% FBS and stimulated with 1 ␮g of E protein/well. Concanavalin A (Con A) and cell culture medium were used as positive and negative controls, respectively. After 48 h, supernatants were harvested and stored at −70 ◦ C. The concentration of interleukin (IL)-2, IL-4, IL-5, TNF-␣ and IFN-␥ in cell supernatants from re-stimulated cells was analyzed using cytometric bead array (CBA) assay (BD Biosciences, Belgium) with a detection limit of 20 pg/ml, according to the manufacturer’s instructions. For IL-4 detection, an enhanced sensitivity kit was used to increase the sensitivity to 0.27 pg/ml. Fluorescence measurements were performed using a FACSCanto A flow cytometer (BD Biosciences) and data were analyzed using the FCAP Array software (BD Biosciences). 2.6. ELISpot The number of IFN-␥ producing cells in spleen was analyzed using Enzyme-Linked ImmunoSpot (ELISpot, Mabtech, Sweden) according to the manufacturer’s instructions. Single cell suspensions, prepared as described [24], were seeded out on filter plates

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Fig. 1. Humoral and cellular immune responses after vaccination with Matrix-MTM adjuvanted WNV E. Six mice per group were immunized with 3 or 10 ␮g of E adjuvanted with 10 ␮g Matrix-MTM at days 0 and 28. IgG1 and IgG2a anti-E titers in serum were determined at days 21 (A) and 42 (B) by an ELISA with E protein. The levels of serum neutralizing antibodies were determined by PRNT90 at day 42 (C). Splenocytes from four mice/group immunized with E (10 ␮g), with or without Matrix-MTM , were restimulated with E and the secretion of IL-2, IFN-␥, TNF-␣ and IL-5 in cell supernatant was analyzed (D). Data is shown as mean ± SEM. For serological data Kruskal–Wallis with adjustment for multiple comparisons followed by the Mann–Whitney test was used to investigate significant statistical differences and for the cytokine data the Mann–Whitney test was used. Significant differences are indicated in the figure where appropriate.

at 0.25 × 106 or 0.125 × 106 cells/well in RPMI medium with 10% FBS and stimulated with 0.5 ␮g E/well. Con A and cell medium were used as positive and negative controls, respectively. Triplicate samples were incubated for 20 h at 37 ◦ C and spots were developed as described by the manufacturer and analyzed using an AID ELR02 ELISpot reader (Autoimmune Diagnostika GmbH, Strassberg, Germany).

levels (27–48 fold, PRNT90, p = 0.017 and p = 0.008) when MatrixMTM adjuvant was used for both antigen doses tested (Fig. 1C). To investigate T cell antigen-specific responses, splenocytes were stimulated ex vivo with E protein two weeks after boosting. The addition of Matrix-MTM adjuvant enhanced the secretion of IL-2, IL-5, TNF-␣ and IFN-␥ after incubation of cells with E protein compared to mice immunized with protein alone (Fig. 1D).

2.7. Statistical analysis

3.2. A strong booster effect is seen by Matrix-MTM adjuvanted WNV E protein

Serological and cytokine data were analyzed with the nonparametric Kruskal–Wallis test or the Mann–Whitney test, adjusted for multiple comparisons by Dunn’s posttest when applicable, and the actual p-value retrieved using Mann–Whitney. All tests were performed using the GraphPad Prism software (GraphPad Prism 5.01 for Windows, GraphPad Software, San Diego, California, USA). 3. Results 3.1. Matrix-MTM increases humoral and cellular responses Mice were immunized s.c. using 10 ␮g of Matrix-MTM in combination with either 3 or 10 ␮g of E or 10 ␮g E alone at days 0 and 28. WNV E protein elicited an antibody response at days 21 and 42 that was enhanced in mice vaccinated with Matrix-MTM adjuvanted protein (Fig. 1A and B). While IgG1 was the dominant isotype after boosting (day 42) with unadjuvanted E protein, there were comparably higher IgG2a antibody titers in the groups receiving Matrix-MTM adjuvanted antigen, which was significant for the group receiving 10 ␮g of Matrix-MTM adjuvanted antigen compared to mice immunized with only E protein (p = 0.0043, Fig. 1B). Immunization with E protein induced neutralizing antibodies against WNV, which accumulated to substantially higher

To assess whether booster vaccinations enhance the antiviral immune response, 12 mice per group were immunized with 3 ␮g of E protein adjuvanted with or without 10 ␮g Matrix-MTM . At day 28 after the primary injection, six mice from each group were boosted and 6 mice were not. At day 42, the mean titer of serum from boosted mice, with either E alone or E adjuvanted with Matrix-MTM , had significantly higher E protein-specific IgG1 levels compared to the groups that were not boosted (61–154 fold, p = 0.0022 and p = 0.0022, Fig. 2). The mean IgG1 anti-E titer of boosted Matrix-MTM adjuvanted E mice was also significantly higher compared to the non-boosted E protein immunized group (p = 0.0022). Only mice immunized with Matrix-MTM adjuvanted E had higher antigenspecific IgG2a titers after boosting compared to the non-boosted comparator group (81-fold, p = 0.0022, Fig. 2). The mean IgG2a titer for the Matrix-MTM adjuvanted E group was also significantly higher after boosting compared to the boosted and non-boosted E protein-only immunized groups (p = 0.0022 and p = 0.0022, Fig. 2). 3.3. Matrix-MTM reduces the antigen dose and increases the durability of the antigen-specific immune response To study the dose requirements of E protein and Matrix-MTM , as well as the durability of the immune response, 8 mice per group

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Fig. 2. Effect of a booster immunization on the humoral response. Twelve mice per group were immunized with 3 ␮g of E adjuvanted with or without 10 ␮g Matrix-MTM . On day 28, six mice from each group were boosted with the same dose of E with or without adjuvant. IgG1 and IgG2a anti-E titers were determined for all mice on day 42 using an ELISA against E protein. Data is shown as mean ± SEM and Kruskal–Wallis with adjustment for multiple comparisons followed by the Mann–Whitney test was used to investigate any significant statistical differences. Significant differences are indicated in the figure where appropriate.

were immunized with 0.5, 1 or 3 ␮g of E adjuvanted with 5 or 10 ␮g Matrix-MTM at days 0 and 28. At day 21 mice immunized with 3 ␮g E adjuvanted with 10, or 5 ␮g Matrix-MTM had higher IgG1 and IgG2a titers (3–23 fold) compared to mice immunized with 3 ␮g E alone, 0.5 ␮g E adjuvanted with 5 or 10 ␮g Matrix-MTM and/or 1 ␮g E adjuvanted with 5 ␮g Matrix-MTM (p = 0.0298–0.0025, Fig. 3A). At day 42, mice immunized with 3 ␮g antigen adjuvanted with 5 or 10 ␮g Matrix-MTM had higher IgG1 titers compared to mice immunized with E alone (p = 0.0003 and p = 0.0006, Fig. 3B) and higher IgG2a levels (p = 0.0009 and p = 0.0009, Fig. 3B) compared to the antigen control. The group immunized with 3 ␮g E adjuvanted with 10 ␮g Matrix-MTM had also significantly higher IgG2a titers compared to the group immunized with 0.5 ␮g E adjuvanted with 5 ␮g Matrix-MTM (p = 0.0006). Notably, the group immunized with only 1 ␮g E adjuvanted with 10 ␮g Matrix-MTM had significantly higher IgG2a titers compared to the unadjuvanted antigen control (p = 0.009, Fig. 3B). Four mice per group were housed for 214 days and the serum antibody levels were studied at days 105 and 186 after boosting. The serum anti-E IgG1- and IgG2a levels were durable and remained elevated at these time points in adjuvanted groups, although they were slightly lower than at day 42 (Fig. 3C). The neutralizing activity of the serum antibodies at day 42 was dependent on the dose of protein and adjuvant, with only the highest antigen dose, 3 ␮g E, adjuvanted with either 5 or 10 ␮g Matrix-MTM generating significantly higher PRNT50 and −90 titers compared to E alone (5–40 fold, p = 0.0026–0.0065, Fig. 3D). In addition, the mice immunized with 3 ␮g E, adjuvanted with either 5 or 10 ␮g Matrix-MTM , also generated significantly higher PRNT50 and −90 titers compared to mice immunized with the lowest dose of antigen and adjuvant, i.e. 0.5 ␮g E adjuvanted with 5 ␮g MatrixMTM (p = 0.0008–0.0028, Fig. 3D). The effect of Matrix-MTM adjuvant on the cellular immune response also was analyzed in context of dose-sparing and durability. At day 42, mice immunized with 3 ␮g of E adjuvanted with 10 ␮g Matrix-MTM had more IFN-␥ producing splenocytes (7.2 fold, p = 0.0286) compared to the unadjuvanted control (Fig. 4A). For the lower antigen dose groups adjuvanted with Matrix-MTM there was a trend of higher numbers of IFN-␥ producing splenocytes, although this did not attain statistical significance. While all mice immunized with Matrix-MTM adjuvanted vaccine produced more IFN-␥, IL-2, IL-5 and IL-4 compared to the unadjuvanted antigen control at day 42, this also failed to reach statistical significance (Fig. 4B). At day 214, splenocytes from mice immunized with 0.5 ␮g of E adjuvanted with 10 ␮g Matrix-MTM produced higher IL-4 and IL-5 levels compared to the control group (p = 0.0286 and p = 0.0286, Fig. 4C).

3.4. Matrix-MTM adjuvanted WNV E induces a balanced and stronger Th1 and Th2 response compared to Alum adjuvanted WNV E To compare Matrix-M with other adjuvant or vaccine preparations, mice were immunized with 3 ␮g E alone, 3 ␮g E adjuvanted with 10 ␮g Matrix-MTM or 1% Alum, or with 1/10 of a horse dose of the veterinary vaccine Duvaxyn® WNV as described in [33]. The mean titer of serum from mice vaccinated with Matrix-MTM adjuvanted E had significantly higher IgG1 anti-E levels compared to only E protein immunized mice and Duvaxyn® immunized mice two weeks after boosting (p = 0.0002 and p = 0.0002, Fig. 5A). Also the Alum adjuvanted E immunized group had significantly elevated IgG1 titers compared to the Duvaxyn® immunized mice (p = 0.0003). Further, Matrix-MTM adjuvanted E also induced significantly higher IgG2a levels compared to Alum adjuvanted E and E alone after boosting (p = 0.0002 and p = 0.0002, Fig. 5A). Only Matrix-MTM adjuvanted E elicited significantly increased levels of neutralizing antibodies (p = 0.0025) compared to antigen alone two weeks after boosting (Fig. 5B). Antigen-specific re-stimulation of splenocytes two weeks after booster immunization revealed that mice vaccinated with Matrix-MTM adjuvanted E had greater secretion of IL-2, IFN-␥, IL-4 and IL-5 compared to only E, alum adjuvanted E and/or Duvaxyn® WNV (Fig. 5C). 3.5. E protein adjuvanted with Matrix-MTM protects against lineage 1 and 2 WNV challenge To study the protective effect of Matrix-MTM adjuvanted E vaccine two independent challenge studies were performed. First, BALB/c mice were vaccinated with 3 or 10 ␮g E adjuvanted with 10 ␮g Matrix-MTM at days 0 and 28 and then challenged i.p. at day 42 with the WNV lineage 1 ITA 09-strain. As a positive control, mice were immunized with the formalin-inactivated WNV virion veterinary vaccine, Duvaxyn® . Mice immunized with low and high E protein doses showed significant protection (p < 0.0001), with survival rates of 90% and 100%, respectively (Fig. 6A). Among the control mice injected with adjuvant only, 8 out of 10 developed signs of severe neuroinvasive disease and were euthanized according to institutional guidelines. There were no differences in the disease score, weight, or protection between the Matrix-MTM adjuvanted E groups and Duvaxyn® WNV (Fig. 6B and C). With the exception of a single animal in the group vaccinated with 3 ␮g E adjuvanted with Matrix-MTM , mice vaccinated with E protein and then challenged with infectious virus showed no clinical signs of

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Fig. 3. Effects of Matrix-MTM on antigen dose sparing and durability of the humoral immune response. Eight mice per group were vaccinated with 0.5, 1 or 3 ␮g of E adjuvanted with 5 or 10 ␮g Matrix-MTM on days 0 and 28. Control mice were immunized with 3 ␮g of E protein. IgG1 and IgG2a anti-E titers were determined for all mice on day 21 (A) and 42 (B) using an ELISA against E protein. IgG1 and IgG2a anti-E titers also were determined on days 133 and 214 after the primary vaccination (C). Serum from all mice on day 42 was analyzed for WNV neutralization using PRNT50 and 90 (D). Data is shown as mean ± SEM and Kruskal–Wallis with adjustment for multiple comparisons followed by the Mann–Whitney test was used to investigate any significant statistical differences. Significant differences are indicated in the figure where appropriate.

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disease and did not become viremic (data not shown). The one immunized animal in the group vaccinated with 3 ␮g E adjuvanted with Matrix-MTM that succumbed to WNV challenge, in retrospective analysis, failed to develop antibody titers after vaccination (Fig. 6D). Finally, the high dose of E protein adjuvanted with MatrixMTM induced significantly higher levels of IgG1 and IgG2a anti-E in serum compared to Duvaxyn® WNV after booster vaccination (p = 0.0001 and p = 0.0002, Fig. 6D) at day 38, four days prior to challenge. In the second experiment, the same groups were included with addition of one group of mice that received only one dose of 10 ␮g E adjuvanted with 10 ␮g Matrix-MTM at day 0, with the other groups being boosted at day 28. At day 42 the mice were challenged i.p. with 105 PFU of WNV lineage 2 AUT 08-strain, which is heterologous to the immunizing E protein. All vaccinated groups showed 100% protection and all control mice developed severe neuroinvasive disease symptoms and were euthanized (Fig. 6E). No differences in disease score or weight between the Matrix-MTM adjuvanted E groups and the Duvaxyn® WNV control group were detected (Fig. 6F and G). Mice vaccinated twice with 3 or 10 ␮g of E protein adjuvanted with Matrix-MTM induced significantly higher levels of anti-E protein IgG1 and IgG2a in serum compared to Duvaxyn® WNV (p = 0.0002–0.0014) and compared to mice vaccinated only once with 10 ␮g E adjuvanted with Matrix-MTM (p = 0.0002 and p = 0.0002) at day 38 post primary immunization, 10 days after booster (Fig. 6H). Overall, our results show that Matrix-MTM adjuvanted E vaccine protects susceptible mice against virulent infection by lineage 1 and 2 WNV strains and that protection already is achieved after a single immunization.

4. Discussion

Fig. 4. Matrix-MTM adjuvanted WNV E protein stimulates a durable cellular immune response at low antigen doses. Splenocytes from four mice per group vaccinated with 0.5, 1 or 3 ␮g of E adjuvanted with 5 or 10 ␮g Matrix-MTM on days 0 and 28 were stimulated with recombinant E protein on days 42 and 214. On day 42, the number of IFN-␥ producing splenocytes in response to antigen-specific re-stimulation was determined by ELISPOT assay (A). The amount of IL-2, IFN-␥, IL-4 and IL-5 secreted in cell supernatant in response to antigen-specific re-stimulation was determined on days 42 (B) and 214 (C) after primary vaccination. Data is shown as mean ± SEM and Kruskal–Wallis with adjustment for multiple comparisons followed by the Mann–Whitney test was used to investigate any significant statistical differences. Significant differences are indicated in the figure where appropriate.

In this paper, we describe the immunogenicity and efficacy of a subunit-based WNV vaccine formulated with bacteriallyexpressed recombinant WNV E protein and the particulate saponin-based adjuvant, Matrix-MTM . The vaccine was protective when immunized mice were challenged with WNV strains belonging either to lineage 1 or 2 that are currently circulating in Europe. The addition of adjuvant Matrix-MTM to the E protein augmented the humoral and cellular immune responses compared to antigen alone and dose sparing and durability effects were observed. The effect of the adjuvant Matrix-MTM compared favorably to Alum, the standard adjuvant of the industry, and Duvaxyn® WNV, an unpurified, formalin-inactivated WNV particle vaccine used for veterinary applications but not suitable for human use. Whereas Alum and Matrix-MTM adjuvanted E induced similar IgG1 anti-E titers in serum, Matrix-MTM induced higher IgG2a anti-E levels and neutralizing antibody titers. Matrix-MTM adjuvanted E also produced higher antigen-specific T cell responses compared to Alum adjuvanted vaccine and Duvaxyn® WNV, with greater secretion of Th1and Th2-type cytokines. Remarkably, the immune response generated by Matrix-MTM adjuvanted E vaccine protected against lethal challenge even when only a single immunization was given. Previously, another E protein vaccine candidate produced in bacteria also showed protection against WNV infection in mice. However, that antigen was generated as a fusion protein, a higher (20 ␮g) dose was required, and the vaccine was adjuvanted with Freund’s complete adjuvant, which is not suitable for human use [18]. The ectodomain of WNV E also has been expressed in insect cells and used in vaccination studies, either adjuvanted with alum or Iscomatrix [11,12,34]. Although similar levels of protection were reported, the antibody response was skewed toward IgG1, whereas the Matrix-MTM adjuvanted E vaccine presented here induced balanced IgG1- and IgG2a-antibody responses. In mice, WNV-specific IgG2a responses are desirable as it has been shown that murine

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Fig. 5. Th1- and Th2-immune responses after immunization with Matrix-MTM , or Alum adjuvanted WNV E vaccine compared to inactivated Duvaxyn® WNV. Eight mice per group were immunized with 3 ␮g of E alone, 3 ␮g of E adjuvanted with 10 ␮g Matrix-MTM or 1% Alum or with 1/10 of a horse dose of Duvaxyn® WNV on days 0 and 28. The IgG1 and IgG2a anti-E titers were analyzed at day 42 (A). Neutralizing activity of serum was assessed using an inhibition of a standard TCID50 assay (NT50), and was performed on samples at day 42 post primary immunization (B). Five mice per vaccination group were re-stimulated with E protein at day 42 after primary vaccination. The amount of IL-2, IFN-␥, IL-4 and IL-5 secretion after antigen-specific splenocyte stimulation was analyzed by CBA (C). Data is shown as mean ± SEM and Kruskal–Wallis with adjustment for multiple comparisons followed by the Mann–Whitney test was used to investigate any significant statistical differences. Significant differences are indicated in the figure where appropriate.

IgG2 isotypes have enhanced effector functions with respect to having increased C1q binding by the antibody Fc region [35]. This property of IgG2a reduced the stoichiometric threshold for neutralization of WNV and enhanced protection in vivo. Hence, compared to other inactivated or subunit based WNV vaccines in development, Matrix-MTM adjuvanted E produce a balanced Th1/Th2 response with regard to both humoral and cellular immunity; this may be desirable as a strong antibody and cellular immune response against WNV is important for protection against infection [36,37]. However, of course our data is only suggestive of CD8 T cell responses, and as no tools for measuring proper CD8 T cell responses are yet available in BALB/c mice, we have to await definitive studies to answer this question when immunodominant H-2d WNV E peptides have been identified. Another potential advantage of WNV E protein expressed in bacteria compared to insect cell-based systems is its more cost effective production. Prior studies have shown that serum from WNV infected patients contained antibodies reactive against E protein that are generated as part of subunit-based vaccines [18]. These results were confirmed by others [38], and imply that isolated E protein

may be a valid target for a WNV vaccine in humans. However, a key unanswered question is whether the antibody repertoire generated in humans after E protein immunization will be similar to that elicited by mice. In the context of natural infection of mice with live, replicating WNV neutralizing antibodies recognize a dominant epitope on the lateral ridge of DIII of the E protein. Humans, however, appear to generate neutralizing antibodies of this specificity less frequently and instead may preferentially recognize a quarternary epitope at the DI-DII hinge interface, which is present only on the surface of the virion and not on the isolated E protein [39,40]. Clinical trials will be required to define the neutralizing and epitope specificity of serum antibodies elicited by our Matrix-MTM adjuvanted E vaccine. By combining a bacterially expressed WNV E protein with the dose-sparing and immune enhancing vaccine adjuvant MatrixMTM , which already has proven safe in human clinical studies [24,25,28], we developed a vaccine candidate that is immunogenic and should be safe (compared to replicating live attenuated vaccine candidates) in at-risk elderly and immunocompromised populations. Futures studies are needed to determine whether

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Fig. 6. Homologous and heterologous WNV challenge of mice after vaccination. Mice (n = 9–10) were challenged on day 42 with 0.2 ml of 104 PFU of WNV ITA-09 (homologous lineage 1) after vaccination at days 0 and 28 with 3 or 10 ␮g of E adjuvanted with 10 ␮g Matrix-MTM or 1/10 of a horse dose of inactivated Duvaxyn® WNV. The survival rate (A), disease score (B) and body weight (C) up to 20 days after challenge is shown. IgG1 and IgG2a anti-E titers at day 38 after primary vaccination were determined by ELISA (D). Mice (n = 8–10) were challenged on day 42 with 0.2 ml of 105 PFU of WNV AUT-08 (heterologous lineage 2) after vaccination with one or two injections of 10 ␮g E adjuvanted with 10 ␮g Matrix-MTM or two injections of 3 ␮g WNV E adjuvanted with 10 ␮g Matrix-MTM or 1/10 of a horse dose of inactivated Duvaxyn® WNV at days 0 and 28. The survival rate (E), disease score (F) and body weight (G) up to 20 days after challenge is shown. IgG1 and IgG2a anti-E titers at day 38 after primary vaccination were determined by ELISA (H). Data is shown as mean ± SEM and Kruskal–Wallis with adjustment for multiple comparisons followed by the Mann–Whitney test was used to investigate any significant statistical differences. Significant differences are indicated in the figure where appropriate.

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