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Preclinical development of a dengue tetravalent recombinant subunit vaccine: Immunogenicity and protective efficacy in nonhuman primates Dhanasekaran Govindarajan a , Steven Meschino a , Liming Guan a , David E. Clements b , Jan H. ter Meulen c , Danilo R. Casimiro a , Beth-Ann G. Coller a , Andrew J. Bett a,∗ a
Merck Research Laboratories, Merck & Co., Inc., Kenilworth, NJ, USA Hawaii Biotech Inc., 99-193 Aiea Heights Drive, Suite 200, Aiea, HI 96701, USA c Immune Design Corporation, 1616 Eastlake Avenue East #310, Seattle, WA 98102, USA b
a r t i c l e
i n f o
Article history: Received 7 April 2015 Received in revised form 6 June 2015 Accepted 14 June 2015 Available online xxx Keywords: Dengue Envelope Subunit vaccine Immunogenicity Efficacy
a b s t r a c t We describe here the preclinical development of a dengue vaccine composed of recombinant subunit carboxy-truncated envelope (E) proteins (DEN-80E) for each of the four dengue serotypes. Immunogenicity and protective efficacy studies in Rhesus monkeys were conducted to evaluate monovalent and tetravalent DEN-80E vaccines formulated with ISCOMATRIXTM adjuvant. Three different doses and two dosing regimens (0, 1, 2 months and 0, 1, 2, and 6 months) were evaluated in these studies. We first evaluated monomeric (DEN4-80E) and dimeric (DEN4-80EZip) versions of DEN4-80E, the latter generated in an attempt to improve immunogenicity. The two antigens, evaluated at 6, 20 and 100 g/dose formulated with ISCOMATRIXTM adjuvant, were equally immunogenic. A group immunized with 20 g DEN4-80E and AlhydrogelTM induced much weaker responses. When challenged with wild-type dengue type 4 virus, all animals in the 6 and 20 g groups and all but one in the DEN4-80EZip 100 g group were protected from viremia. Two out of three monkeys in the AlhydrogelTM group had breakthrough viremia. A similar study was conducted to evaluate tetravalent formulations at low (3, 3, 3, 6 g of DEN1-80E, DEN2-80E, DEN3-80E and DEN4-80E respectively), medium (10, 10, 10, 20 g) and high (50, 50, 50, 100 g) doses. All doses were comparably immunogenic and induced high titer, balanced neutralizing antibodies against all four DENV. Upon challenge with the four wild-type DENV, all animals in the low and medium dose groups were protected against viremia while two animals in the high-dose group exhibited breakthrough viremia. Our studies also indicated that a 0, 1, 2 and 6 month vaccination schedule is superior to the 0, 1, and 2 month schedule in terms of durability. Overall, the subunit vaccine was demonstrated to induce strong neutralization titers resulting in protection against viremia following challenge even 8-12 months after the last vaccine dose. © 2015 Elsevier Ltd. All rights reserved.
1. Introduction Dengue is the most important mosquito-borne viral illnesses of the tropics and the subtropics, with significant morbidity and mortality [1–4]. Recent studies suggest that an estimated 4 billion people around the globe are at risk of infection with approximately 390 million infections occurring worldwide annually, of which there are about 96 million symptomatic dengue cases [5,6]. Though mainly a disease of the tropics and subtropics, dengue is rapidly
∗ Corresponding author at: Mail Stop WP14-2, 770 Sumneytown Pike, West Point, PA 19486, USA. Tel.: +1 215 652 3673; fax: +1 215 652 2142. E-mail address: andrew [email protected] (A.J. Bett).
becoming a global public health concern due to the spread of the mosquito vector, increased urbanization, international commerce and global air travel. Dengue virus belongs to the Flavivirus genus in the family Flaviviridae [7]. There are four serotypes of dengue virus (DENV-1, DENV-2, DENV-3 and DENV-4), which are transmitted by Aedes sp. of mosquitoes. Infection with any of the virus serotypes ranges from asymptomatic infection to flu-like illness (dengue fever [DF]), and rarely to the severe life-threatening forms of dengue hemorrhagic fever and dengue shock syndrome (DHF/DSS) [8,9]. Generally, it is believed that infection with a single serotype of dengue leads to lifelong protection against that particular serotype, but not the other serotypes. However, when an individual previously exposed to one serotype is infected at a later date with a different serotype, they are
http://dx.doi.org/10.1016/j.vaccine.2015.06.067 0264-410X/© 2015 Elsevier Ltd. All rights reserved.
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at increased risk of developing the severe forms of illness, DHF and DSS. Due to the existence of four serotypes and the complex nature of the illness, it is believed that a dengue vaccine should be tetravalent in nature so that it will provide robust protective immunity against all four serotypes simultaneously and not sensitize people for DHF/DSS [10,11]. Currently, there are no vaccines or antiviral therapies available for dengue and the only line of treatment is supportive therapy [12]. Given the expanding global burden of disease, there is an increased need for a dengue vaccine. Several efforts have been underway over the past 70 years toward the development of a dengue vaccine. Vaccine candidates at various stages of development include live attenuated viruses (LAV), inactivated viruses, protein subunit vaccines, vectored vaccines, and DNA vaccines [13–15]. Moreover, each of the vaccine concepts is associated with its own advantages and disadvantages in terms of safety, immunogenicity, and feasibility of developing into a product. The most advanced clinical candidate, a tetravalent vaccine consisting of recombinant chimeric yellow fever LAV expressing the prM and E genes of each of the four DENV serotypes (CYD), has recently completed Phase 3 clinical trials [16–18]. Tetravalent LAV formulations have a history of and the potential for viral interaction/interference resulting in the failure to generate balanced tetravalent immunity/balanced protection. The modest efficacy reported for the CYD vaccine in Phase II and III trials (especially for flavivirus naïve subjects and against DENV2, Sanofi trial) further emphasizes that there are practical challenges for attaining a balanced protective responses with LAV vaccines [10,16–19]. Furthermore, the prolonged three-dose regimen (0, 6 and 12 months) used for the CYD LAV presents challenges for implementation and compliance. Recombinant subunit vaccines offer several potential advantages compared to LAV approaches. These include, (1) improved safety in populations where LAV’s are often not used (e.g. the very young, very old and immune compromised); (2) they are not subject to the issue of viral interference and the level of each component can be easily adjusted to target balanced tetravalent immune responses; (3) they offer the potential for an accelerated dosage regimen which can lead to greater vaccine compliance and suitability for travelers, military personnel and use during outbreaks [20]. However, non-replicating vaccines such as recombinant subunits may have some limitations. These include a higher cost of goods, immune responses that may encompass more limited epitope coverage or be of shorter duration, and a reduced ability to induce cell mediated immune responses compared to live vaccines. However, these limitations may be overcome at least in part by the addition of adjuvants. Therefore in order to capitalize on the advantages of non-replicating vaccines while limiting the disadvantages we are currently developing a tetravalent recombinant subunit based dengue vaccine in combination with ISCOMATRIXTM adjuvant. In this paper, we describe the preclinical development of a tetravalent formulation of a recombinant subunit vaccine containing a carboxy-truncated envelope (E) protein (DEN-80E) of each of the four dengue serotypes.
2. Materials and methods 2.1. Cells and viruses Vero cells (ATCC CCL-81) were maintained in Medium 199 supplemented with 10% heat-inactivated fetal bovine serum (HyClone), glutamine (Mediatech) and penicillin-streptomycin (Mediatech). These cells were used for virus growth, virus titration and virus neutralization assays. Stably transformed Drosophila
S2 cells that were used to express the DEN-80E subunit proteins of the four dengue viruses have been described elsewhere in detail [21]. The viruses used in the assays described here were DENV1 (strain WestPac-74), DENV-2 (strain S18603), DENV-3 (strain CH53489), and DENV-4 (strain TVP-360). All the viruses were kindly provided by Alan Barrett (University of Texas Medical Branch, Galveston, TX). Viruses were amplified in Vero cells cultured at 37 ◦ C at a low multiplicity of infection. Briefly, Vero cells grown in 225 cm2 flasks were infected with 0.01 multiplicity of infection of each of the DENV subtypes. Supernatant from each of the infected cultures was harvested at 5 days post-infection, clarified at 1000 × g for 10 min, divided into 0.2–0.5 mL aliquots, flash-frozen on dry ice, and stored at −70 ◦ C. All virus aliquots were thawed on ice immediately prior to use in all assays. 2.2. Dengue envelope (E) proteins The DEN-80E proteins belonging to all four DENV serotypes (DENV-1 strain 258848, DENV-2 strain PR159 S1, DENV-3 strain CH53489, and DENV-4 strain H241) were expressed in Drosophila S2 cells [21]. Briefly, dengue sequences encoding the full-length prM protein and 80% of the E protein (truncation at amino acid 395 for DENV-1, DENV-2 and DENV-4, 393 for DENV-3) were individually cloned into the Drosophila expression vector pMttXho and then transformed into Drosophila S2 cells. The carboxy terminal truncations remove the carboxy stem and transmembrane region of the E protein and result in secretion of DEN-80E with native-like biological structure into the cell culture medium. The secreted recombinant DEN-80E subunits were purified from clarified cell culture medium by immunoaffinity chromatography using a conformationally sensitive monoclonal antibody (4G2) [22]. The purified bulk biologic substances were formulated in phosphate buffered saline at pH 7.2 and filtered into single-use bags and stored at −20 ◦ C. DEN4-80EZip is a dimeric version of the DENV-4 envelope protein (derived from H241 stain). It consists of the DEN4-80E protein joined to a C-terminal flexible linker and leucine zipper domain to facilitate dimerization. The dimeric version of DEN4-80E was generated as a potential mechanism to improve the immunogenicity of the monomeric DEN4-80E which had been noted to induce slightly lower virus neutralizing antibody titers in previous preclinical studies [21]. To generate the DEN4-80EZip expression plasmid, the DEN4-80E monomer-expressing plasmid was modified to include a leucine zipper region fused in frame at the carboxy terminus of the DEN4-80E sequence to allow for the expression of a DEN480EZip dimer product. The third to last codon in the leucine zipper sequence was designed as a cysteine to allow for the two monomers which comprise the dimer to be linked via a disulfide bond. The disulfide linked DEN4-80E leucine zipper dimer is referred to as DEN4-80EZip. The method of production of DEN4-80EZip is essentially the same as that of the other DEN-80E proteins (see above) with the exception that the purified biological substance is formulated in 20 mM glycine pH 9.0. Since DEN4-80EZip and DEN4-80E are based on the same amino acid sequence expressed in the same expression system and react with conformationally sensitive monoclonal antibodies in the same way it is presumed that the structures are similar. However, neither the monomeric nor the dimeric form of DEN4-80E have been analyzed by X-ray crystallography so the exact structures have not been confirmed. 2.3. Rhesus monkey studies All animal studies were performed at the New Iberia Primate Research Center (New Iberia, LA) according to the Institutional Animal Care and Use Committee-approved protocols. Healthy adult,
Please cite this article in press as: Govindarajan D, et al. Preclinical development of a dengue tetravalent recombinant subunit vaccine: Immunogenicity and protective efficacy in nonhuman primates. Vaccine (2015), http://dx.doi.org/10.1016/j.vaccine.2015.06.067
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Table 1 Schedule and formulations used in rhesus immunogenicity and challenge studies. Group
Monkeys per group
Formulation
Schedule (months)
Challenge virus serotype (3 monkeys per type) Month 8
TM
90 ISCO
TM
0, 1, 2
units ISCOMATRIX
Month 14
1
12
DENV1, DENV2 DENV3, DENV4
2
3
100 g DEN4-80E 90 ISCOTM units ISCOMATRIXTM
0, 1, 2
3
3
20 g DEN4-80E 90 ISCOTM units ISCOMATRIXTM
0, 1, 2
4
3
6 g DEN4-80E 90 ISCOTM units ISCOMATRIXTM
0, 1, 2, 6
DENV4
5
3
100 g DEN4-80EZip 90 ISCOTM units ISCOMATRIXTM
0, 1, 2
DENV4
6
3
20 g DEN4-80EZip 90 ISCOTM units ISCOMATRIXTM
0, 1, 2
7
3
6 g DEN4-80EZip 90 ISCOTM units ISCOMATRIXTM
0, 1, 2, 6
8
3
20 g DEN4-80E 225 g AlhydrogelTM †
0, 1, 2
9
12
50 g DEN1-80E 50 g DEN2-80E 50 g DEN3-80E 100 g DEN4-80E 90 ISCOTM units ISCOMATRIXTM
0, 1, 2
10
12
10 g DEN1-80E 10 g DEN2-80E 10 g DEN3-80E 20 g DEN4-80E 90 ISCOTM units ISCOMATRIXTM
0, 1, 2
11
12
3 g DEN1-80E 3 g DEN2-80E 3 g DEN3-80E 6 g DEN4-80E 90 ISCOTM units ISCOMATRIXTM
0, 1, 2, 6
12
12
No vaccine control
DENV4 DENV4
DENV4 DENV4 DENV4 DENV1, DENV2 DENV3, DENV4
DENV1, DENV2 DENV3, DENV4
DENV1, DENV2 DENV3, DENV4
DEN1, DEN2 DEN3, DEN4
† AlhydrogelTM dose represents the amount of elemental aluminum. ISCOMATRIXTM : ISCOMATRIXTM adjuvant.
Indian rhesus macaques of either sex, weighing more than 3 kg, and which were flavivirus (DENV-1, 2, 3 and 4, and West Nile) antibodynegative by ELISA were utilized in this study. Three monkeys per group were used when evaluating monovalent DEN4-80E vaccines and 12 monkeys per group were used to evaluate the tetravalent formulations or the ISCOMATRIXTM adjuvant negative control group. ISCOMATRIXTM adjuvant (ISCOMATRIX; CSL Biotherapies Inc., King of Prussia, PA, USA; ISCOMATRIX is a registered trademark of ISCOTEC Ab a CSL company; ISCO is a registered trademark of CSL) was prepared as described previously [23] and used at 90 ISCOTM Units/dose. The vaccination schedules are described in Table 1. The monovalent DENV antigens, DEN4-80E and DEN480EZip, were evaluated at low, medium, and high doses (6, 20 and 100 g/dose respectively). Similarly the tetravalent formulations were evaluated at low (3, 3, 3, 6 g of DEN1-80E, DEN2-80E, DEN3-80E and DEN4-80E respectively) medium (10, 10, 10, 20 g) and high (50, 50, 50, 100 g) doses. Formulations further comprised ISCOMATRIXTM adjuvant at a dose of 90 ISCOTM units or AlhydrogelTM at a dose of 225 g of elemental aluminum as outlined in Table 1. The medium- and high-dose groups received three doses of vaccine at 0, 1, and 2 months and the low-dose group received four doses of vaccine at 0, 1, 2 and 6 months. Each 0.5-mL dose was administered by intramuscular inoculation. After vaccination, the animals were observed daily for any changes at the
inoculation site or other changes in activity or feeding habits that might indicate an adverse reaction to the vaccine. Blood samples from the immunized animals were collected at monthly intervals and used in an infra-red dye (IRD)-based neutralization assays to evaluate immunogenicity of the various antigen formulations. After receiving a complete vaccination series, each animal was challenged by subcutaneous injection of 5 log10 plaque forming units (PFU) of one of 4 near wild-type viruses as outlined in Table 1. The medium-dose groups were challenged at study month 8 (6 months after the last dose of vaccine), while the high- and lowdose groups were challenged at study month 14 (12 and 8 months after the last vaccine dose, respectively). The challenge times were selected in order to assess vaccine efficacy relatively late after vaccination to more closely mimic actual field conditions. The monkeys in the groups receiving monovalent DEN4-80E vaccines were challenged with DENV-4 (strain TVP-360), while the monkeys in groups receiving tetravalent vaccine formulations were randomized into 4 groups of 3 animals each and challenged with DENV-1 (strain WestPac-74), DENV-2 (strain S16803), DENV-3 (strain CH53489), or DENV-4 (strain TVP-360). All these challenge viruses except DENV-3 are heterologous viruses compared to the vaccine components and differ from the DEN-80E proteins in our vaccine immunogens by 9 (DENV-1), 11 (DENV-2) and 8 (DENV-4) amino acids in the E protein. These viruses have been previously shown to
Please cite this article in press as: Govindarajan D, et al. Preclinical development of a dengue tetravalent recombinant subunit vaccine: Immunogenicity and protective efficacy in nonhuman primates. Vaccine (2015), http://dx.doi.org/10.1016/j.vaccine.2015.06.067
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cause viremia of significant duration in dengue virus naïve-rhesus macaques (data not shown) and belong to a panel of viruses widely used by reference laboratories and vaccine developers [24]. There is no morbidity or mortality expected upon challenge in this model. Animals were bled daily for 10 days post-challenge for viremia assessments. Blood samples were also collected from all animals on the day of challenge (i.e. prior to virus challenge) to determine the virus neutralization titer on the day of challenge. Group mean days of viremia were calculated by dividing the total number of viremic days in a single group divided by the number of animals in that group [25]. 2.4. Plaque assay Plaque assays to measure the virus titers for challenge viruses were performed on 6-well plates containing confluent Vero cells. The monolayer was incubated with 10-fold serial virus dilutions for 1 h at 37 ◦ C. After virus adsorption, the inoculum was removed and replaced with 2–3 mL of Medium 199 containing 2% fetal bovine serum and 1.0% methyl cellulose (Sigma), and the cells were incubated at 37 ◦ C. After 5 days of incubation, the methyl cellulose overlays were removed and the cells were fixed and stained with 80% methanol/crystal violet solution for 2 h at room temperature. Following fixing and staining, the plates were washed and allowed to dry. Viral plaques were counted from the dried plates to determine virus titers and reported as PFU. 2.5. Infra-red dye dengue neutralization assay An infra-red dye (IRD) based high-throughput and sensitive neutralization assay was developed and utilized for all virus neutralization assays. Briefly, in a 96-well plate heat-inactivated serum samples were serially diluted (2-fold, from 1:10) in 50 L of 2% medium 199 followed by the addition of 50 PFU in 50 L of virus, and incubated at 37 ◦ C for 1 h. Following neutralization, the entire mixture was added onto Vero cells previously seeded in 96-well plates and incubated at 37 ◦ C. After 4 days of incubation, the culture media from the plates was removed and the cells were fixed with 3.7% formaldehyde in PBS for 30 min and permeabilized by washing twice with 200 L 0.1% Triton X-100/PBS. All washing and dispensing steps were automated using the BioTek® EL406 plate washer system (Winooski, VT). Immunostaining of the plates was carried out using a pan-flavivirus primary antibody 4G2 ([22]; 2.8 g/mL) as the primary antibody and a biotinylated horse antimouse IgG (Vector Laboratories, CA, 7.5 g/mL) as the secondary antibody. The secondary antibody was followed by the addition of a cocktail of IRDye® 800CW Streptavidin (1:1000, LiCor Biosciences) and DRAQ5 (1:10,000, Shepshed, Leicestershire, UK). The DNA stain DRAQ5 was used to stain Vero cell DNA for normalization in the IRD assay. Following 1 h incubation, the mixture was removed and the plates were washed, air-dried and scanned using an infrared Odyssey® Sa imaging system (Li-Cor Biosciences). Serum end-point neutralization titers (averaged from duplicate wells) were defined as the reciprocal of the highest serum dilution that reduces the 800 nm/700 nm fluorescence integrated intensity ratio greater than 50% when compared to virus control included on each assay plate. If a sample failed to neutralize at this dilution, a titer of 1:5 was assigned to that sample and this dilution was used to calculate the geometric mean of that group. 2.6. Viremia assays Blood samples (∼5 mL per animal) were collected daily from each of the dengue virus-challenged monkeys over a period of 10 days post challenge. Serum was separated from the blood samples and stored in aliquots at −80 ◦ C until assayed. Viremia assays on
the serum samples were performed as per the method described previously [26] with some modifications. Briefly, about 250 L of each serum sample was inoculated onto Vero cells in T-25 flasks. Following incubation at 37 ◦ C for 7 days, duplicate aliquots of supernatant from each culture flask were subjected to an IRDbased immunostaining viremia assay. The IRD viremia assay used is identical to the IRD Dengue neutralization assay described above, except that the sample inoculated onto the cells was the previously amplified serum (day 7 cell culture supernatant). This assay was chosen over conventional plaque assay due to its increased sensitivity, specificity and higher-throughput nature. Wells that gave an 800 nm/700 nm ratio greater than twice that of the cell control wells were considered positive for virus.
3. Results 3.1. Immunogenicity and protective efficacy of monovalent DEN4-80E The DEN4-80E antigen formulated with ISCOMATRIXTM adjuvant has been shown to induce a virus neutralizing antibody response when administered as monovalent or tetravalent formulations both in mice and in non-human primates [21]. However, it was observed that the DEN4-80E component induced lower antibody response than the other three components when given at the same dose and there was breakthrough viremia in a monkey vaccinated with a 5 g dose of DEN4-80E upon challenge with DENV4. In an attempt to improve the immunogenicity of the DEN4 component, a dimeric form of DEN4-80E was constructed. This dimeric form, DEN4-80EZip, was then compared to the monomeric form in a monkey immunogenicity and efficacy study. Groups of 3 monkeys were immunized with low, medium and high (6, 20 and 100 g, respectively) doses of monomeric DEN4-80E or dimeric DEN480EZip subunits adjuvanted with ISCOMATRIXTM adjuvant at 90 ISCOTM units. A negative control group was included that received ISCOMATRIXTM adjuvant only, at 90 ISCOTM units per dose. For comparative purposes, an additional group was included in the study that received the medium-dose of DEN4-80E (20 g) formulated with 225 g of elemental aluminum (AlhydrogelTM ). The medium and high dose groups were immunized at 0, 1 and 2 months while the low dose groups received an additional dose at 6 months. The magnitude and the durability of DENV-4 virus-specific neutralizing antibodies in the vaccinated animals were determined every 1 to 2 months from baseline (study week 0) through study week 60 using the LiCor-based microneutralization assay (Fig. 1). Two doses of the vaccine were required for a detectable neutralizing antibody response in most vaccinated animals. The third immunization administered at study month 2 resulted in a modest increase in the geometric mean (GMT) neutralizing titers. The GMT measured at study month 3 (1 month post dose 3) for all groups to all four dengue types are summarized in Table 2. The GMT at 1 month post-dose 3 for DEN4-80E at the low, medium and high doses were 508, 508, and 320 respectively, while the titers for DEN4-80EZip were 640, 1016, and 320. As shown in Table 2 and Fig. 1, DEN4-80E and DEN4-80EZip formulated with 90 ISCOTM units of ISCOMATRIXTM adjuvant were comparably immunogenic across the antigen doses evaluated, although there was a slight trend for higher/more durable responses with DEN480EZip when administered at a low antigen dose (6 g) compared to the monomeric DEN4-80E at the same dose. The neutralizing antibody responses in monkeys given the monovalent DEN4-80E or DEN4-80EZip formulations were largely serotype specific as evidenced by low levels of cross-reactive neutralizing antibodies against the other dengue virus serotypes (Table 2). The durability of the responses was generally comparable between DEN4-80E and
Please cite this article in press as: Govindarajan D, et al. Preclinical development of a dengue tetravalent recombinant subunit vaccine: Immunogenicity and protective efficacy in nonhuman primates. Vaccine (2015), http://dx.doi.org/10.1016/j.vaccine.2015.06.067
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Fig. 1. Longitudinal geometric mean virus neutralization titers in rhesus monkeys immunized with various DEN4-80E vaccine formulations. Groups of 3 monkeys were immunized with high (100 g, panel A), medium (20 g, panel B) and low (6 g, panel C) doses of the monovalent DEN4-80E or dimeric DEN4-80Ezip adjuvanted with 90 ISCOTM units of ISCOMATRIXTM adjuvant. A negative control group was included that received ISCOMATRIXTM adjuvant only, at 90 ISCOTM units per dose (Saline/IMX). All groups were immunized at 0, 1 and 2 months while the low dose group was given an additional dose at 6 month (blue arrows). The ISCOMATRIXTM adjuvant only and medium dose groups were challenged at month 8 with near wild type-dengue 4 virus while the high and low dose groups were challenged at month 14 (red arrows). A naïve control group was included at month 14 for the challenge. Virus neutralizing antibody titers were measured over a period of 15 months using a LiCor-based neutralization assay. The geometric mean virus neutralization titers to DENV4 for each formulation at each time point are presented. ISCOMATRIXTM : ISCOMATRIXTM . (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
DEN4-80EZip with responses slowly declining following the third vaccine dose. Mid dose DEN-80E formulated with ISCOMATRIXTM adjuvant was found to result in substantially higher neutralizing GMT (508) than AlhydrogelTM formulated DEN4-80E (32). The superiority of ISCOMATRIXTM adjuvant over AlhydrogelTM was observed previously in studies evaluating the DEN2-80E subunit vaccine [21]. The booster vaccination given to the low dose groups at 6 months resulted in a good anamnestic response that boosted neutralization titers to levels equivalent or higher than those seen one month post dose 3, but then slowly declined thereafter. The protective efficacy of the monomeric and dimeric DEN4-80E as single component subunit vaccines was evaluated by challenging the vaccinated animals with 105 PFU of live DENV4 strain TVP-360 administered subcutaneously. Viremia was assessed in blood samples collected daily for 10 days post challenge using a highly sensitive and virus-specific infra-red dye-based immunodetection assay. Animals in the adjuvant only control group and the medium-dose groups were challenged at 8 months (i.e. 6 months
post dose 3). The viremia measurements in these animals are shown in Table 3. Upon challenge with DENV4 strain TVP-360, all control animals became viremic, whereas the animals vaccinated with the medium dose of monomeric (DEN4-80E) or dimeric (DEN480EZip) vaccines formulated with ISCOMATRIXTM adjuvant did not show any viremia. The adjuvant only control-immunized animals exhibited a group mean of 3 days of viremia. One control monkey (ID-A7E050) was viremic for 1 day while the remaining two animals were viremic for 3 and 5 days. Viremia ranging between 2 and 3 days (mean of 1.67 days) was detected in 2 out of the 3 animals that received the medium-dose of DEN4-80E formulated with 225 g of elemental aluminum (AlhydrogelTM , group 8). There was a positive correlation between lack of viremia upon challenge and higher DENV4-specific virus neutralization titer on the day of challenge. All the animals in groups 1 and 8 had undetectable (≤1:10) virus neutralization titers on the day of challenge. Interestingly, one animal (ID-A7E039) in the DEN4-80E adjuvanted with AlhydrogelTM group was protected from viremia even though it did
Please cite this article in press as: Govindarajan D, et al. Preclinical development of a dengue tetravalent recombinant subunit vaccine: Immunogenicity and protective efficacy in nonhuman primates. Vaccine (2015), http://dx.doi.org/10.1016/j.vaccine.2015.06.067
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Table 2 DENV4-specific neutralizing antibody titers (LiCor50 GMT) induced by recombinant monovalent (DEN4-80E and DEN4-80EZip) subunit vaccines at study month 3 (1 month post dose 3). Group
Monkeys per group
Formulation
LiCor50 titers (GMT) Anti-DENV-1
TM
90 ISCO
TM
units ISCOMATRIX
Anti-DENV-2
Anti-DENV-3
Anti-DENV-4
1
12
5
5
5
5
2
3
100 g DEN4-80E 90 ISCOTM units ISCOMATRIXTM
25
20
16
320
3
3
20 g DEN4-80E 90 ISCOTM units ISCOMATRIXTM
32
16
25
508
4
3
6 g DEN4-80E 90 ISCOTM units ISCOMATRIXTM
16
13
20
508
5
3
100 g DEN4-80EZip 90 ISCOTM units ISCOMATRIXTM
63
32
25
320
6
3
20 g DEN4-80Ezip 90 ISCOTM units ISCOMATRIXTM
63
25
32
1016
7
3
6 g DEN4-80Ezip 90 ISCOTM units ISCOMATRIXTM
63
32
25
640
8
3
20 g DEN4-80E 225 g AlhydrogelTM †
6
5
5
32
† AlhydrogelTM dose represents the amount of elemental aluminum. LiCor50 result of
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Contents lists available at ScienceDirect
Vaccine journal homepage: www.elsevier.com/locate/vaccine
Preclinical development of a dengue tetravalent recombinant subunit vaccine: Immunogenicity and protective efficacy in nonhuman primates Dhanasekaran Govindarajan a , Steven Meschino a , Liming Guan a , David E. Clements b , Jan H. ter Meulen c , Danilo R. Casimiro a , Beth-Ann G. Coller a , Andrew J. Bett a,∗ a
Merck Research Laboratories, Merck & Co., Inc., Kenilworth, NJ, USA Hawaii Biotech Inc., 99-193 Aiea Heights Drive, Suite 200, Aiea, HI 96701, USA c Immune Design Corporation, 1616 Eastlake Avenue East #310, Seattle, WA 98102, USA b
a r t i c l e
i n f o
Article history: Received 7 April 2015 Received in revised form 6 June 2015 Accepted 14 June 2015 Available online xxx Keywords: Dengue Envelope Subunit vaccine Immunogenicity Efficacy
a b s t r a c t We describe here the preclinical development of a dengue vaccine composed of recombinant subunit carboxy-truncated envelope (E) proteins (DEN-80E) for each of the four dengue serotypes. Immunogenicity and protective efficacy studies in Rhesus monkeys were conducted to evaluate monovalent and tetravalent DEN-80E vaccines formulated with ISCOMATRIXTM adjuvant. Three different doses and two dosing regimens (0, 1, 2 months and 0, 1, 2, and 6 months) were evaluated in these studies. We first evaluated monomeric (DEN4-80E) and dimeric (DEN4-80EZip) versions of DEN4-80E, the latter generated in an attempt to improve immunogenicity. The two antigens, evaluated at 6, 20 and 100 g/dose formulated with ISCOMATRIXTM adjuvant, were equally immunogenic. A group immunized with 20 g DEN4-80E and AlhydrogelTM induced much weaker responses. When challenged with wild-type dengue type 4 virus, all animals in the 6 and 20 g groups and all but one in the DEN4-80EZip 100 g group were protected from viremia. Two out of three monkeys in the AlhydrogelTM group had breakthrough viremia. A similar study was conducted to evaluate tetravalent formulations at low (3, 3, 3, 6 g of DEN1-80E, DEN2-80E, DEN3-80E and DEN4-80E respectively), medium (10, 10, 10, 20 g) and high (50, 50, 50, 100 g) doses. All doses were comparably immunogenic and induced high titer, balanced neutralizing antibodies against all four DENV. Upon challenge with the four wild-type DENV, all animals in the low and medium dose groups were protected against viremia while two animals in the high-dose group exhibited breakthrough viremia. Our studies also indicated that a 0, 1, 2 and 6 month vaccination schedule is superior to the 0, 1, and 2 month schedule in terms of durability. Overall, the subunit vaccine was demonstrated to induce strong neutralization titers resulting in protection against viremia following challenge even 8-12 months after the last vaccine dose. © 2015 Elsevier Ltd. All rights reserved.
1. Introduction Dengue is the most important mosquito-borne viral illnesses of the tropics and the subtropics, with significant morbidity and mortality [1–4]. Recent studies suggest that an estimated 4 billion people around the globe are at risk of infection with approximately 390 million infections occurring worldwide annually, of which there are about 96 million symptomatic dengue cases [5,6]. Though mainly a disease of the tropics and subtropics, dengue is rapidly
∗ Corresponding author at: Mail Stop WP14-2, 770 Sumneytown Pike, West Point, PA 19486, USA. Tel.: +1 215 652 3673; fax: +1 215 652 2142. E-mail address: andrew [email protected] (A.J. Bett).
becoming a global public health concern due to the spread of the mosquito vector, increased urbanization, international commerce and global air travel. Dengue virus belongs to the Flavivirus genus in the family Flaviviridae [7]. There are four serotypes of dengue virus (DENV-1, DENV-2, DENV-3 and DENV-4), which are transmitted by Aedes sp. of mosquitoes. Infection with any of the virus serotypes ranges from asymptomatic infection to flu-like illness (dengue fever [DF]), and rarely to the severe life-threatening forms of dengue hemorrhagic fever and dengue shock syndrome (DHF/DSS) [8,9]. Generally, it is believed that infection with a single serotype of dengue leads to lifelong protection against that particular serotype, but not the other serotypes. However, when an individual previously exposed to one serotype is infected at a later date with a different serotype, they are
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at increased risk of developing the severe forms of illness, DHF and DSS. Due to the existence of four serotypes and the complex nature of the illness, it is believed that a dengue vaccine should be tetravalent in nature so that it will provide robust protective immunity against all four serotypes simultaneously and not sensitize people for DHF/DSS [10,11]. Currently, there are no vaccines or antiviral therapies available for dengue and the only line of treatment is supportive therapy [12]. Given the expanding global burden of disease, there is an increased need for a dengue vaccine. Several efforts have been underway over the past 70 years toward the development of a dengue vaccine. Vaccine candidates at various stages of development include live attenuated viruses (LAV), inactivated viruses, protein subunit vaccines, vectored vaccines, and DNA vaccines [13–15]. Moreover, each of the vaccine concepts is associated with its own advantages and disadvantages in terms of safety, immunogenicity, and feasibility of developing into a product. The most advanced clinical candidate, a tetravalent vaccine consisting of recombinant chimeric yellow fever LAV expressing the prM and E genes of each of the four DENV serotypes (CYD), has recently completed Phase 3 clinical trials [16–18]. Tetravalent LAV formulations have a history of and the potential for viral interaction/interference resulting in the failure to generate balanced tetravalent immunity/balanced protection. The modest efficacy reported for the CYD vaccine in Phase II and III trials (especially for flavivirus naïve subjects and against DENV2, Sanofi trial) further emphasizes that there are practical challenges for attaining a balanced protective responses with LAV vaccines [10,16–19]. Furthermore, the prolonged three-dose regimen (0, 6 and 12 months) used for the CYD LAV presents challenges for implementation and compliance. Recombinant subunit vaccines offer several potential advantages compared to LAV approaches. These include, (1) improved safety in populations where LAV’s are often not used (e.g. the very young, very old and immune compromised); (2) they are not subject to the issue of viral interference and the level of each component can be easily adjusted to target balanced tetravalent immune responses; (3) they offer the potential for an accelerated dosage regimen which can lead to greater vaccine compliance and suitability for travelers, military personnel and use during outbreaks [20]. However, non-replicating vaccines such as recombinant subunits may have some limitations. These include a higher cost of goods, immune responses that may encompass more limited epitope coverage or be of shorter duration, and a reduced ability to induce cell mediated immune responses compared to live vaccines. However, these limitations may be overcome at least in part by the addition of adjuvants. Therefore in order to capitalize on the advantages of non-replicating vaccines while limiting the disadvantages we are currently developing a tetravalent recombinant subunit based dengue vaccine in combination with ISCOMATRIXTM adjuvant. In this paper, we describe the preclinical development of a tetravalent formulation of a recombinant subunit vaccine containing a carboxy-truncated envelope (E) protein (DEN-80E) of each of the four dengue serotypes.
2. Materials and methods 2.1. Cells and viruses Vero cells (ATCC CCL-81) were maintained in Medium 199 supplemented with 10% heat-inactivated fetal bovine serum (HyClone), glutamine (Mediatech) and penicillin-streptomycin (Mediatech). These cells were used for virus growth, virus titration and virus neutralization assays. Stably transformed Drosophila
S2 cells that were used to express the DEN-80E subunit proteins of the four dengue viruses have been described elsewhere in detail [21]. The viruses used in the assays described here were DENV1 (strain WestPac-74), DENV-2 (strain S18603), DENV-3 (strain CH53489), and DENV-4 (strain TVP-360). All the viruses were kindly provided by Alan Barrett (University of Texas Medical Branch, Galveston, TX). Viruses were amplified in Vero cells cultured at 37 ◦ C at a low multiplicity of infection. Briefly, Vero cells grown in 225 cm2 flasks were infected with 0.01 multiplicity of infection of each of the DENV subtypes. Supernatant from each of the infected cultures was harvested at 5 days post-infection, clarified at 1000 × g for 10 min, divided into 0.2–0.5 mL aliquots, flash-frozen on dry ice, and stored at −70 ◦ C. All virus aliquots were thawed on ice immediately prior to use in all assays. 2.2. Dengue envelope (E) proteins The DEN-80E proteins belonging to all four DENV serotypes (DENV-1 strain 258848, DENV-2 strain PR159 S1, DENV-3 strain CH53489, and DENV-4 strain H241) were expressed in Drosophila S2 cells [21]. Briefly, dengue sequences encoding the full-length prM protein and 80% of the E protein (truncation at amino acid 395 for DENV-1, DENV-2 and DENV-4, 393 for DENV-3) were individually cloned into the Drosophila expression vector pMttXho and then transformed into Drosophila S2 cells. The carboxy terminal truncations remove the carboxy stem and transmembrane region of the E protein and result in secretion of DEN-80E with native-like biological structure into the cell culture medium. The secreted recombinant DEN-80E subunits were purified from clarified cell culture medium by immunoaffinity chromatography using a conformationally sensitive monoclonal antibody (4G2) [22]. The purified bulk biologic substances were formulated in phosphate buffered saline at pH 7.2 and filtered into single-use bags and stored at −20 ◦ C. DEN4-80EZip is a dimeric version of the DENV-4 envelope protein (derived from H241 stain). It consists of the DEN4-80E protein joined to a C-terminal flexible linker and leucine zipper domain to facilitate dimerization. The dimeric version of DEN4-80E was generated as a potential mechanism to improve the immunogenicity of the monomeric DEN4-80E which had been noted to induce slightly lower virus neutralizing antibody titers in previous preclinical studies [21]. To generate the DEN4-80EZip expression plasmid, the DEN4-80E monomer-expressing plasmid was modified to include a leucine zipper region fused in frame at the carboxy terminus of the DEN4-80E sequence to allow for the expression of a DEN480EZip dimer product. The third to last codon in the leucine zipper sequence was designed as a cysteine to allow for the two monomers which comprise the dimer to be linked via a disulfide bond. The disulfide linked DEN4-80E leucine zipper dimer is referred to as DEN4-80EZip. The method of production of DEN4-80EZip is essentially the same as that of the other DEN-80E proteins (see above) with the exception that the purified biological substance is formulated in 20 mM glycine pH 9.0. Since DEN4-80EZip and DEN4-80E are based on the same amino acid sequence expressed in the same expression system and react with conformationally sensitive monoclonal antibodies in the same way it is presumed that the structures are similar. However, neither the monomeric nor the dimeric form of DEN4-80E have been analyzed by X-ray crystallography so the exact structures have not been confirmed. 2.3. Rhesus monkey studies All animal studies were performed at the New Iberia Primate Research Center (New Iberia, LA) according to the Institutional Animal Care and Use Committee-approved protocols. Healthy adult,
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Table 1 Schedule and formulations used in rhesus immunogenicity and challenge studies. Group
Monkeys per group
Formulation
Schedule (months)
Challenge virus serotype (3 monkeys per type) Month 8
TM
90 ISCO
TM
0, 1, 2
units ISCOMATRIX
Month 14
1
12
DENV1, DENV2 DENV3, DENV4
2
3
100 g DEN4-80E 90 ISCOTM units ISCOMATRIXTM
0, 1, 2
3
3
20 g DEN4-80E 90 ISCOTM units ISCOMATRIXTM
0, 1, 2
4
3
6 g DEN4-80E 90 ISCOTM units ISCOMATRIXTM
0, 1, 2, 6
DENV4
5
3
100 g DEN4-80EZip 90 ISCOTM units ISCOMATRIXTM
0, 1, 2
DENV4
6
3
20 g DEN4-80EZip 90 ISCOTM units ISCOMATRIXTM
0, 1, 2
7
3
6 g DEN4-80EZip 90 ISCOTM units ISCOMATRIXTM
0, 1, 2, 6
8
3
20 g DEN4-80E 225 g AlhydrogelTM †
0, 1, 2
9
12
50 g DEN1-80E 50 g DEN2-80E 50 g DEN3-80E 100 g DEN4-80E 90 ISCOTM units ISCOMATRIXTM
0, 1, 2
10
12
10 g DEN1-80E 10 g DEN2-80E 10 g DEN3-80E 20 g DEN4-80E 90 ISCOTM units ISCOMATRIXTM
0, 1, 2
11
12
3 g DEN1-80E 3 g DEN2-80E 3 g DEN3-80E 6 g DEN4-80E 90 ISCOTM units ISCOMATRIXTM
0, 1, 2, 6
12
12
No vaccine control
DENV4 DENV4
DENV4 DENV4 DENV4 DENV1, DENV2 DENV3, DENV4
DENV1, DENV2 DENV3, DENV4
DENV1, DENV2 DENV3, DENV4
DEN1, DEN2 DEN3, DEN4
† AlhydrogelTM dose represents the amount of elemental aluminum. ISCOMATRIXTM : ISCOMATRIXTM adjuvant.
Indian rhesus macaques of either sex, weighing more than 3 kg, and which were flavivirus (DENV-1, 2, 3 and 4, and West Nile) antibodynegative by ELISA were utilized in this study. Three monkeys per group were used when evaluating monovalent DEN4-80E vaccines and 12 monkeys per group were used to evaluate the tetravalent formulations or the ISCOMATRIXTM adjuvant negative control group. ISCOMATRIXTM adjuvant (ISCOMATRIX; CSL Biotherapies Inc., King of Prussia, PA, USA; ISCOMATRIX is a registered trademark of ISCOTEC Ab a CSL company; ISCO is a registered trademark of CSL) was prepared as described previously [23] and used at 90 ISCOTM Units/dose. The vaccination schedules are described in Table 1. The monovalent DENV antigens, DEN4-80E and DEN480EZip, were evaluated at low, medium, and high doses (6, 20 and 100 g/dose respectively). Similarly the tetravalent formulations were evaluated at low (3, 3, 3, 6 g of DEN1-80E, DEN2-80E, DEN3-80E and DEN4-80E respectively) medium (10, 10, 10, 20 g) and high (50, 50, 50, 100 g) doses. Formulations further comprised ISCOMATRIXTM adjuvant at a dose of 90 ISCOTM units or AlhydrogelTM at a dose of 225 g of elemental aluminum as outlined in Table 1. The medium- and high-dose groups received three doses of vaccine at 0, 1, and 2 months and the low-dose group received four doses of vaccine at 0, 1, 2 and 6 months. Each 0.5-mL dose was administered by intramuscular inoculation. After vaccination, the animals were observed daily for any changes at the
inoculation site or other changes in activity or feeding habits that might indicate an adverse reaction to the vaccine. Blood samples from the immunized animals were collected at monthly intervals and used in an infra-red dye (IRD)-based neutralization assays to evaluate immunogenicity of the various antigen formulations. After receiving a complete vaccination series, each animal was challenged by subcutaneous injection of 5 log10 plaque forming units (PFU) of one of 4 near wild-type viruses as outlined in Table 1. The medium-dose groups were challenged at study month 8 (6 months after the last dose of vaccine), while the high- and lowdose groups were challenged at study month 14 (12 and 8 months after the last vaccine dose, respectively). The challenge times were selected in order to assess vaccine efficacy relatively late after vaccination to more closely mimic actual field conditions. The monkeys in the groups receiving monovalent DEN4-80E vaccines were challenged with DENV-4 (strain TVP-360), while the monkeys in groups receiving tetravalent vaccine formulations were randomized into 4 groups of 3 animals each and challenged with DENV-1 (strain WestPac-74), DENV-2 (strain S16803), DENV-3 (strain CH53489), or DENV-4 (strain TVP-360). All these challenge viruses except DENV-3 are heterologous viruses compared to the vaccine components and differ from the DEN-80E proteins in our vaccine immunogens by 9 (DENV-1), 11 (DENV-2) and 8 (DENV-4) amino acids in the E protein. These viruses have been previously shown to
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cause viremia of significant duration in dengue virus naïve-rhesus macaques (data not shown) and belong to a panel of viruses widely used by reference laboratories and vaccine developers [24]. There is no morbidity or mortality expected upon challenge in this model. Animals were bled daily for 10 days post-challenge for viremia assessments. Blood samples were also collected from all animals on the day of challenge (i.e. prior to virus challenge) to determine the virus neutralization titer on the day of challenge. Group mean days of viremia were calculated by dividing the total number of viremic days in a single group divided by the number of animals in that group [25]. 2.4. Plaque assay Plaque assays to measure the virus titers for challenge viruses were performed on 6-well plates containing confluent Vero cells. The monolayer was incubated with 10-fold serial virus dilutions for 1 h at 37 ◦ C. After virus adsorption, the inoculum was removed and replaced with 2–3 mL of Medium 199 containing 2% fetal bovine serum and 1.0% methyl cellulose (Sigma), and the cells were incubated at 37 ◦ C. After 5 days of incubation, the methyl cellulose overlays were removed and the cells were fixed and stained with 80% methanol/crystal violet solution for 2 h at room temperature. Following fixing and staining, the plates were washed and allowed to dry. Viral plaques were counted from the dried plates to determine virus titers and reported as PFU. 2.5. Infra-red dye dengue neutralization assay An infra-red dye (IRD) based high-throughput and sensitive neutralization assay was developed and utilized for all virus neutralization assays. Briefly, in a 96-well plate heat-inactivated serum samples were serially diluted (2-fold, from 1:10) in 50 L of 2% medium 199 followed by the addition of 50 PFU in 50 L of virus, and incubated at 37 ◦ C for 1 h. Following neutralization, the entire mixture was added onto Vero cells previously seeded in 96-well plates and incubated at 37 ◦ C. After 4 days of incubation, the culture media from the plates was removed and the cells were fixed with 3.7% formaldehyde in PBS for 30 min and permeabilized by washing twice with 200 L 0.1% Triton X-100/PBS. All washing and dispensing steps were automated using the BioTek® EL406 plate washer system (Winooski, VT). Immunostaining of the plates was carried out using a pan-flavivirus primary antibody 4G2 ([22]; 2.8 g/mL) as the primary antibody and a biotinylated horse antimouse IgG (Vector Laboratories, CA, 7.5 g/mL) as the secondary antibody. The secondary antibody was followed by the addition of a cocktail of IRDye® 800CW Streptavidin (1:1000, LiCor Biosciences) and DRAQ5 (1:10,000, Shepshed, Leicestershire, UK). The DNA stain DRAQ5 was used to stain Vero cell DNA for normalization in the IRD assay. Following 1 h incubation, the mixture was removed and the plates were washed, air-dried and scanned using an infrared Odyssey® Sa imaging system (Li-Cor Biosciences). Serum end-point neutralization titers (averaged from duplicate wells) were defined as the reciprocal of the highest serum dilution that reduces the 800 nm/700 nm fluorescence integrated intensity ratio greater than 50% when compared to virus control included on each assay plate. If a sample failed to neutralize at this dilution, a titer of 1:5 was assigned to that sample and this dilution was used to calculate the geometric mean of that group. 2.6. Viremia assays Blood samples (∼5 mL per animal) were collected daily from each of the dengue virus-challenged monkeys over a period of 10 days post challenge. Serum was separated from the blood samples and stored in aliquots at −80 ◦ C until assayed. Viremia assays on
the serum samples were performed as per the method described previously [26] with some modifications. Briefly, about 250 L of each serum sample was inoculated onto Vero cells in T-25 flasks. Following incubation at 37 ◦ C for 7 days, duplicate aliquots of supernatant from each culture flask were subjected to an IRDbased immunostaining viremia assay. The IRD viremia assay used is identical to the IRD Dengue neutralization assay described above, except that the sample inoculated onto the cells was the previously amplified serum (day 7 cell culture supernatant). This assay was chosen over conventional plaque assay due to its increased sensitivity, specificity and higher-throughput nature. Wells that gave an 800 nm/700 nm ratio greater than twice that of the cell control wells were considered positive for virus.
3. Results 3.1. Immunogenicity and protective efficacy of monovalent DEN4-80E The DEN4-80E antigen formulated with ISCOMATRIXTM adjuvant has been shown to induce a virus neutralizing antibody response when administered as monovalent or tetravalent formulations both in mice and in non-human primates [21]. However, it was observed that the DEN4-80E component induced lower antibody response than the other three components when given at the same dose and there was breakthrough viremia in a monkey vaccinated with a 5 g dose of DEN4-80E upon challenge with DENV4. In an attempt to improve the immunogenicity of the DEN4 component, a dimeric form of DEN4-80E was constructed. This dimeric form, DEN4-80EZip, was then compared to the monomeric form in a monkey immunogenicity and efficacy study. Groups of 3 monkeys were immunized with low, medium and high (6, 20 and 100 g, respectively) doses of monomeric DEN4-80E or dimeric DEN480EZip subunits adjuvanted with ISCOMATRIXTM adjuvant at 90 ISCOTM units. A negative control group was included that received ISCOMATRIXTM adjuvant only, at 90 ISCOTM units per dose. For comparative purposes, an additional group was included in the study that received the medium-dose of DEN4-80E (20 g) formulated with 225 g of elemental aluminum (AlhydrogelTM ). The medium and high dose groups were immunized at 0, 1 and 2 months while the low dose groups received an additional dose at 6 months. The magnitude and the durability of DENV-4 virus-specific neutralizing antibodies in the vaccinated animals were determined every 1 to 2 months from baseline (study week 0) through study week 60 using the LiCor-based microneutralization assay (Fig. 1). Two doses of the vaccine were required for a detectable neutralizing antibody response in most vaccinated animals. The third immunization administered at study month 2 resulted in a modest increase in the geometric mean (GMT) neutralizing titers. The GMT measured at study month 3 (1 month post dose 3) for all groups to all four dengue types are summarized in Table 2. The GMT at 1 month post-dose 3 for DEN4-80E at the low, medium and high doses were 508, 508, and 320 respectively, while the titers for DEN4-80EZip were 640, 1016, and 320. As shown in Table 2 and Fig. 1, DEN4-80E and DEN4-80EZip formulated with 90 ISCOTM units of ISCOMATRIXTM adjuvant were comparably immunogenic across the antigen doses evaluated, although there was a slight trend for higher/more durable responses with DEN480EZip when administered at a low antigen dose (6 g) compared to the monomeric DEN4-80E at the same dose. The neutralizing antibody responses in monkeys given the monovalent DEN4-80E or DEN4-80EZip formulations were largely serotype specific as evidenced by low levels of cross-reactive neutralizing antibodies against the other dengue virus serotypes (Table 2). The durability of the responses was generally comparable between DEN4-80E and
Please cite this article in press as: Govindarajan D, et al. Preclinical development of a dengue tetravalent recombinant subunit vaccine: Immunogenicity and protective efficacy in nonhuman primates. Vaccine (2015), http://dx.doi.org/10.1016/j.vaccine.2015.06.067
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Fig. 1. Longitudinal geometric mean virus neutralization titers in rhesus monkeys immunized with various DEN4-80E vaccine formulations. Groups of 3 monkeys were immunized with high (100 g, panel A), medium (20 g, panel B) and low (6 g, panel C) doses of the monovalent DEN4-80E or dimeric DEN4-80Ezip adjuvanted with 90 ISCOTM units of ISCOMATRIXTM adjuvant. A negative control group was included that received ISCOMATRIXTM adjuvant only, at 90 ISCOTM units per dose (Saline/IMX). All groups were immunized at 0, 1 and 2 months while the low dose group was given an additional dose at 6 month (blue arrows). The ISCOMATRIXTM adjuvant only and medium dose groups were challenged at month 8 with near wild type-dengue 4 virus while the high and low dose groups were challenged at month 14 (red arrows). A naïve control group was included at month 14 for the challenge. Virus neutralizing antibody titers were measured over a period of 15 months using a LiCor-based neutralization assay. The geometric mean virus neutralization titers to DENV4 for each formulation at each time point are presented. ISCOMATRIXTM : ISCOMATRIXTM . (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
DEN4-80EZip with responses slowly declining following the third vaccine dose. Mid dose DEN-80E formulated with ISCOMATRIXTM adjuvant was found to result in substantially higher neutralizing GMT (508) than AlhydrogelTM formulated DEN4-80E (32). The superiority of ISCOMATRIXTM adjuvant over AlhydrogelTM was observed previously in studies evaluating the DEN2-80E subunit vaccine [21]. The booster vaccination given to the low dose groups at 6 months resulted in a good anamnestic response that boosted neutralization titers to levels equivalent or higher than those seen one month post dose 3, but then slowly declined thereafter. The protective efficacy of the monomeric and dimeric DEN4-80E as single component subunit vaccines was evaluated by challenging the vaccinated animals with 105 PFU of live DENV4 strain TVP-360 administered subcutaneously. Viremia was assessed in blood samples collected daily for 10 days post challenge using a highly sensitive and virus-specific infra-red dye-based immunodetection assay. Animals in the adjuvant only control group and the medium-dose groups were challenged at 8 months (i.e. 6 months
post dose 3). The viremia measurements in these animals are shown in Table 3. Upon challenge with DENV4 strain TVP-360, all control animals became viremic, whereas the animals vaccinated with the medium dose of monomeric (DEN4-80E) or dimeric (DEN480EZip) vaccines formulated with ISCOMATRIXTM adjuvant did not show any viremia. The adjuvant only control-immunized animals exhibited a group mean of 3 days of viremia. One control monkey (ID-A7E050) was viremic for 1 day while the remaining two animals were viremic for 3 and 5 days. Viremia ranging between 2 and 3 days (mean of 1.67 days) was detected in 2 out of the 3 animals that received the medium-dose of DEN4-80E formulated with 225 g of elemental aluminum (AlhydrogelTM , group 8). There was a positive correlation between lack of viremia upon challenge and higher DENV4-specific virus neutralization titer on the day of challenge. All the animals in groups 1 and 8 had undetectable (≤1:10) virus neutralization titers on the day of challenge. Interestingly, one animal (ID-A7E039) in the DEN4-80E adjuvanted with AlhydrogelTM group was protected from viremia even though it did
Please cite this article in press as: Govindarajan D, et al. Preclinical development of a dengue tetravalent recombinant subunit vaccine: Immunogenicity and protective efficacy in nonhuman primates. Vaccine (2015), http://dx.doi.org/10.1016/j.vaccine.2015.06.067
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Table 2 DENV4-specific neutralizing antibody titers (LiCor50 GMT) induced by recombinant monovalent (DEN4-80E and DEN4-80EZip) subunit vaccines at study month 3 (1 month post dose 3). Group
Monkeys per group
Formulation
LiCor50 titers (GMT) Anti-DENV-1
TM
90 ISCO
TM
units ISCOMATRIX
Anti-DENV-2
Anti-DENV-3
Anti-DENV-4
1
12
5
5
5
5
2
3
100 g DEN4-80E 90 ISCOTM units ISCOMATRIXTM
25
20
16
320
3
3
20 g DEN4-80E 90 ISCOTM units ISCOMATRIXTM
32
16
25
508
4
3
6 g DEN4-80E 90 ISCOTM units ISCOMATRIXTM
16
13
20
508
5
3
100 g DEN4-80EZip 90 ISCOTM units ISCOMATRIXTM
63
32
25
320
6
3
20 g DEN4-80Ezip 90 ISCOTM units ISCOMATRIXTM
63
25
32
1016
7
3
6 g DEN4-80Ezip 90 ISCOTM units ISCOMATRIXTM
63
32
25
640
8
3
20 g DEN4-80E 225 g AlhydrogelTM †
6
5
5
32
† AlhydrogelTM dose represents the amount of elemental aluminum. LiCor50 result of
