Alum adjuvanted rabies DNA vaccine confers 80% protection against lethal 50 LD50 rabies challenge virus standard strain

Molecular Immunology 85 (2017) 166–173

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Alum adjuvanted rabies DNA vaccine confers 80% protection against lethal 50 LD50 rabies challenge virus standard strain Rajni Garg a,b , Manpreet Kaur a,c,1 , Ankur Saxena a,d,1 , Rajendra Prasad b , Rakesh Bhatnagar a,∗ a

Laboratory of Molecular Biology and Genetic Engineering, School of Biotechnology, Jawaharlal Nehru University, New Delhi, 110067 Delhi, India Amity Institute of Biotechnology, Amity University, Gurgaon (Manesar), 122413 Haryana, India c Vaccine and Infectious Disease Research Center, Translational Health Science and Technology Institute, NCR Biotech Science Cluster, 3rd Milestone, Faridabad-Gurgaon Expressway, Faridabad, 121001 Haryana, India d Fish Health Division, Diagnostic Virology Laboratory, ICAR—Directorate of Coldwater Fisheries Research, Anusandhan Bhawan, Industrial Area, Bhimtal 263136, District Nainital, Uttarakhand, India b

a r t i c l e

i n f o

Article history: Received 5 September 2016 Received in revised form 15 February 2017 Accepted 18 February 2017 Keywords: Encephalitis DNA vaccine Adjuvants Challenge Protective efficacy

a b s t r a c t Rabies is a serious concern world-wide. Despite availability of rabies vaccines for long; their efficacy, safety, availability and cost effectiveness has been a tremendous issue. This calls for improvement of rabies vaccination strategies. DNA vaccination has immense potential in this regard. The DNA vaccine pgp.LAMP-1 conferred 60% protection to BALB/c mice against 20 LD50 rabies challenge virus standard (CVS) strain challenge. Upon supplementation with Emulsigen-D, the vaccine formulation conferred complete protection against lethal challenge. To assess the feasibility of this vaccine formulation for human use, it was tested along with other FDA approved adjuvants, namely, Alum, Immuvac, Montanide ISA720 VG. Enhanced immune response correlated with high IgG antibody titer, Th2 biased response with a high level of rabies virus neutralizing antibodies (RVNAs) and IgG1/IgG2a ratio >1, observed upon alum supplementation of the rabies DNA vaccine. The total IgG antibody titer was 2 IU/ml and total RVNA titer was observed to be 4 IU/ml which is eight times higher than the minimum protective titer recommended by WHO. Furthermore, it conferred 80% protection against challenge with 50 LD50 of the rabies CVS strain, conducted in compliance with the potency test for rabies recommended by the National Institutes of Health (NIH), USA. Previously, we have established pre-clinical safety of this vaccine as per the guidelines of Schedule Y, FDA as well as The European Agency for evaluation of Medicinal Products. The vaccine showed no observable toxicity at the site of injection as well as at systemic level in Wistar rats when administered with 10X recommended dose. Therefore, supplementation of rabies DNA vaccine, pgp.LAMP-1 with alum would lead to development of a non-toxic, efficacious, stable and affordable vaccine that can be used to combat high numbers of fatal rabies infections tormenting developing countries. © 2017 Elsevier Ltd. All rights reserved.

1. Introduction Rabies is a zoonotic disease, caused by rabies virus, a member of the Lyssavirus genus of Rhabdoviridae family. This virus uses mammals like domestic animals (dogs) and wild animals like raccoons, skunks, bats and foxes as its reservoir. The public health burden

Abbreviations: Ab, antibody; Ig, immunoglobulin; gp, glycoprotein; RVNA, rabies virus neutralization antibody; LAMP, 1 lysosomal-associated membrane protein-1. ∗ Corresponding author. E-mail address: [email protected] (R. Bhatnagar). 1 Equally contributing authors. http://dx.doi.org/10.1016/j.molimm.2017.02.011 0161-5890/© 2017 Elsevier Ltd. All rights reserved.

of rabies is huge; approximately 59,000 deaths occur every year (Hampson et al., 2015). WHO advises pre-exposure prophylaxis for high-risk area travelers, personnels occupationally involved with wild animal reservoirs, veterinarians or researchers working on rabies infected animals (WHO, Rabies, 2013). Post-exposure prophylaxis is used for people bitten by rabid animals. Currently, Human Diploid Cell Vaccine (HDCV), Purified Chick Embryo Cell (PCEC) vaccine, Purified Vero Cell Vaccine (PVCV) and Purified Duck Embryo Vaccine (PDEV) are advised by WHO for human usage. HDCV poses several local side effects like erythema in 85% patients, swelling in 61% patients and pruritus in 44% patients upon intradermal booster vaccination (Burridge et al., 1984). The high cost of HDCV is another factor demanding for alternative vaccination

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strategies (Dreesen et al., 1989). The adverse reactions of PVCV are similar but occur at lower frequency with symptoms of fever seen in only 6 of 100 vaccinees (Wang et al., 2000; WHO, 2010). DNA vaccine has emerged as one of the potent alternatives for rabies prevention. The beauty of DNA vaccine lies in the ease and cost-effectiveness of the procedure of generating DNA vaccine. It’s capability to be stored at room temperature during transportation further enhances the scope of its usage in areas with limited cold storage accessibility. Plasmid DNA vaccinations have been used against a variety of infectious diseases including bacterial, viral and parasitic infections (Alves et al., 1998; Kaur et al., 2009; Wang et al., 1998) as they induce persistent cellular and humoral response (Liu, 2013). Novel adjuvants, targeting sequences, enhanced delivery methods, use of epigenetics and prime-boost DNA vaccine strategies have been further used to boost their immunogenicity (Li et al., 2012). Previously, we have shown that DNA vaccine construct pgp.LAMP-1 (glycoprotein G gene with Lysosomal Associated Membrane Protein-1 targeting sequence) conferred partial protection (60%) against intracerebral challenge with 20 LD50 of rabies CVS strain in BALB/c mice (Kaur et al., 2009). To improve its protective efficacy, the vaccine was supplemented with Emulsigen-D which elicited enhanced total antibody as well as Rabies Virus Neutralizing antibody (RVNA) titer than the naked DNA. Further, the improved formulation imparted complete protection upon lethal challenge (Kaur et al., 2010). Our vaccine has been proven to be completely safe as per the guidelines of Schedule Y, FDA as well as The European Agency for evaluation of Medicinal Products (Garg et al., 2014). The vaccine alone as well as in combination with Emulsigen-D, was tested for local and systemic toxicity in Swiss Albino mice as well as Wistar rats and found to be safe even at 10X therapeutic dose (Garg et al., 2014). In the present study, we have explored the effect of novel FDA approved adjuvants on pgp.LAMP-1 vaccination and compared their protective efficacy as per the NIH guidelines in BALB/c mice (Meslin et al., 1996). In addition to Emulsigen-D (MVP Lab. Inc); we investigated other potent and novel adjuvants, namely Alum (Sigma), Immuvac (Cadila Pharmaceuticals Ltd.), Montanide ISA720 VG (Seppic) in order to achieve a highly potent and efficacious vaccine formulation for human usage. Emulsigen-D is an oil-in-water emulsified adjuvant containing DDA immunostimulant which was previously used in our study, where optimized rabies DNA vaccine formulation imparted complete protection against challenge with 20 LD50 of CVS (Kaur et al., 2010). Emulsigen-D has been widely used for adjuvanting DNA vaccines such as in vaccine against Toxoplasma gondii as well as inactivated vaccines like Foot and mouth disease vaccine resulting in a heightened immune response ´ (Hiszczynska-Sawicka et al., 2010; Park et al., 2014). Despite its immense potential in enhancing immune response, Emulsigen-D is still not approved by FDA for human use. This prompted us to assess supplementation of RDV with FDA approved adjuvants so that the vaccine can be improvised for human usage. Amongst the different adjuvants, alum has been most extensively used in vaccines, including human vaccines for more than seven decades. Though, the exact molecular mechanism of its action is still not known, it has been postulated that adsorption to alum leads to increased antigen availability at the site of injection thereby elevating uptake by antigen-presenting cells (APCs) as well as dendritic cells (DCs) in vitro (Hem and Hogenesch, 2007; Morefield et al., 2005). The intraperitoneal administration of alum induced recruitment of monocytes which after internalization of vaccine antigen, migrate to the draining lymph nodes, and differentiate into inflammatory DCs (Kool et al., 2008). However, its inability to induce Th1 antibody isotypes or cellular immune responses, and poor impact on polysaccharide antigens limits its applicability in some scenarios (Asherson, 1995). Immuvac consisting of heat − killed Mycobac-

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terium w (now referred to as Mycobacterium indicus pranii, MIP) has been approved for human use against leprosy, where it resulted increased immune responses to M. leprae antigens and enhanced bacterial clearance in patients (Sharma et al., 2005). Additionally, MIP has been shown to induce protection against tuberculosis (TB) in animal models and early sputum conversion in TB patients (Patel et al., 2002; Singh et al., 1991). Furthermore, it has shown potential for tuberculin conversion and increased CD4+ cell count in human immunodeficiency virus (HIV)-infected people (Nyasulu, 2010). Montanide ISA-720 VG, water-in-squalene emulsion, has been shown to elicit high antibody response in several animal species. It has been evaluated in human vaccine trials in combination with malaria (Lawrence et al., 1997) and HIV vaccines (Toledo et al., 2001). Montanide ISA 720 VG co-administered with Plasmodium vivax circumsporozoite protein based polypeptide was highly immunogenic in BALB/c mice and Aotus monkeys (Arévalo-Herrera et al., 2011). Moreover, it has established its safety profile in various studies for human vaccines (Ascarateil et al., 2015). Its mode of action is proposed to be the formation of a depot at the injection site from where antigen is released slowly (Miles et al., 2005). In the current study, the impact of these FDA approved adjuvants on rabies DNA vaccine pgp.LAMP-1 was assessed in BALB/c mice. Various immune parameters elicited along with protective efficacy imparted were compared in view of developing a highly efficacious, stable and cost effective rabies vaccine. 2. Materials and methods 2.1. Cells, virus, vaccine and mice Baby hamster kidney (BHK)-21 cells were procured from National Centre for Cell Science (NCCS), Pune, India and maintained in Dulbecco’s modified Eagle’s medium (DMEM, Sigma) supplemented with 10% heat-inactivated fetal bovine serum (FBS, GIBCO, Life Technologies) containing 100 U/ml Penicillin (Amersham) and 100 ␮g/ml Streptomycin (Amersham), in a humidified 5% CO2 incubator at 37 ◦ C. Mice brain infected with Rabies Challenge Virus Standard Strain (CVS) was procured from Indian Veterinary Research Institute (IVRI), Izzatnagar, Bareilly. The rabies DNA vaccine encoding glycoprotein G gene of rabies virus (pgp.LAMP-1, GenBank Accession Number EU715588) used in this study was generated by our group (Kaur et al., 2010). Three to four weeks BALB/c mice weighing 20–22 g were obtained from National Centre for Laboratory Animal Sciences, NIN, Hyderabad, India and were maintained in animal holding room of BSL3 laboratory. All the animal experiments were done in compliance with Institutional Animal Ethics committee, Jawaharlal Nehru University and Council for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Ministry of Social Justice and Empowerment, Government of India; as per the Three Rs guidelines of Russell and Burch (http://3rs.ccac.ca/en/about/three-rs.html, Romanian Parliament, 2002). 2.2. Standardization of rabies virus: culture, titration, lethal dose 2.2.1. Rabies Challenge Virus Standard culture in BHK-21 cells At 80% confluence, BHK-21 cells were infected with rabies virus at multiplicity of infection (MOI) range of 0.1–0.5. The infection was allowed to proceed for 60 min at 37 ◦ C with gentle stirring after every 10–15 min. The cells were harvested after 2 days of infection and trypsinized to produce the CVS. The supernatant collected was used to infect fresh BHK-21 cells to get Passage 1 cells (P1). Likewise generated, Passage 5 (P5) BHK-21 cells were trypsinized and the cell supernatant was collected in complete DMEM. The supernatant was centrifuged at 800–1000g for 15 min at 4 ◦ C, aliquoted

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and frozen at −80 ◦ C. The harvest was checked for presence of the virus by mouse inoculation test in which different groups of mice were injected intracerebrally with different dilutions of virus in a dose of 0.03 ml and observed for rabies related symptoms for 14 days. The infective titer of the harvest was established 3 days after it was frozen. 2.2.2. Titration of virus for TCID50 105 BHK-21 cells were added to each well of 96-well microtiter plates. The plates were then incubated for 24 h at 37 ◦ C with 5% CO2 . The medium in the microtiter plates was discarded. Tenfold serial dilutions (10−1 –10−8 ) of the viral suspension was performed in incomplete DMEM and 50 ␮l was added to each well. The plate was then incubated for an hour at 37 ◦ C with 5% CO2 . 200 ␮l of DMEM, containing 5% FBS, was added and incubated for 3 days at 37 ◦ C in 5% CO2 . The cells were then stained using the fluorescently labeled nucleoprotein antibody (Bio-Rad). The wells giving fluorescent signal were considered to be positive. The 50% end point was calculated. The titer calculation was made using the Spearman–Kärber formula (Meslin et al., 1996). The Proportionate Distance (PD) between the two dilutions in between 50% death was calculated as follows: PD = (% next above 50%) – 50%/(% next above 50%) – (% next below 50%). Log10 (lower dilution) i.e. the dilution in which position is next above 50% was calculated and TCID50 is the sum of PD and Log10 (lower dilution). 2.2.3. Rabies Challenge Virus Standard Strain LD50 calculation Six 10-fold dilutions (10−2 –10−7 ) of infected mice brain were prepared in 2% chilled inactivated normal horse serum in distilled water. Each dilution of volume 0.03 ml was administered intracerebrally to six groups of twelve mice each. The groups of mice were kept under observation for development of rabies specific symptoms till 14 days post-inoculation. The numbers of mice dying 5th day onwards were recorded. The virus titer was calculated by Reed & Muench method. The virus titer should not be less than 105 LD50 /0.03 ml. The cumulative deaths from each dilution were calculated starting from the highest to the lowest dilution and percent death from each dilution was calculated. The Percent death was calculated using the formula: Percent death = (Percent positive above 50%–50)/[(Percent positive above 50%)–(Percent positive below 50%)]. LD50 was found to be 105 /30 ␮l. 2.3. Vaccination of mice with different adjuvants For immune analysis and protection elicited by rabies DNA vaccine (RDV) supplemented with Immuvac, Montanide ISA 720 VG, Alum and Emulsigen-D as adjuvants, seven groups of mice (n = 10) were vaccinated each with 100 ␮g of RDV along with respective adjuvant. For mice immunized with Immuvac, 107 mycobacterial cells were used for single immunization. Montanide ISA 720 VG, Alum and Emulsigen-D were used at a concentration of 70%, 20% and 20% v/v respectively. Only RDV was also used. In negative control groups, vector control and healthy control, mice were vaccinated with vector DNA and PBS respectively. All groups of mice were vaccinated intramuscularly and first booster dose was given 14 days after primary immunization and the second booster was given after 28 days of primary immunization. 2.4. Immunological assays 2.4.1. Total IgG titer and reference sera test Blood samples from vaccinated mice were collected by retroorbital puncture on day 13th, 27th and 41st of the first immunization. Serum, thus collected was heat inactivated for 30 min at 56 ◦ C. The immune analysis was performed by determining the total antibody titer, antibody isotyping and Rabies virus

neutralization antibody (RVNA) titers by RFFIT (Meslin et al., 1996). To calculate the antibody titer in terms of international unit (IU/ml), reference sera test was performed and two fold serial dilutions of the test sera and reference sera were made in each well. 100 TCID50 of viral suspension in a volume of 50 ␮l was added to each well, incubated for 30 min and 105 BHK-21 cells were added to it. The plate was then incubated for 72 h at 37 ◦ C in 5% CO2 . The media was then aspirated and the cells were fixed using cold 4% paraformaldehyde. The cells were then treated with 1:200 dilution of FITC- conjugated anti-rabies nucleocaspid antibody (Bio-Rad). The plate was examined under fluorescence microscope (Nikon, Tokyo, Japan) for fluorescent foci. The total antibody titer is the reciprocal of dilution at which 50% of the cells are infected. The 1:32 dilution of the reference serum is considered to neutralize the virus completely and is expressed as 1 IU/ml. Test serum was compared with the reference serum for titration in terms of IU/ml.

2.4.2. IgG isotyping The IgG1 and IgG2a isotypes were measured from the sera of immunized mice at 13th, 27th and 41st day using ELISA as described previously (Kaur et al., 2009). The IgG1 and IgG2a titers are represented as geometric mean of absorbance values (O.D.450 ) obtained from pooled serum samples (in triplicates) from each group.

2.4.3. Determination of Rabies Virus Neutralizing Antibody titer (RVNA) RVNA titers were determined using rapid fluorescent foci inhibition test (RFFIT), from sera obtained on 13th, 27th and 41st day as test and equine hyper-immune anti rabies serum diluted to contain 32 IU/ml as reference serum. Samples were assayed in triplicate in two-fold serial dilutions starting from 1:2 to 1:128. 100 TCID50 of the virus was added and after incubation, 105 BHK 21 cells were added to each well. The serum neutralization end-point titer is defined as the dilution factor of the highest serum dilution at which 50% of the observed microscopic fields contain one or more infected cells. RVNA titer is expressed in International units/ml (IU/ml).

2.4.4. Secreted cytokines estimation after in-vitro stimulation of splenocytes The spleens were isolated from sacrificed mice of different groups and they were ground in between the frosted slides to obtain single cell suspension. RBCs were lysed using RBC lysis buffer (Sigma, St. Louis, MO, USA). The splenocytes were washed with DMEM and resuspended in complete DMEM containing 10−6 M ␤-mercaptoethanol. Viability of the cells was assessed by trypan blue exclusion test. The cells were seeded at a concentration of 106 in each well of a 24-well tissue culture plate (Corning Inc., NY, USA). The cells were stimulated without antigen, with 5 ␮g/ml ␤-propiolactone (BPL) inactivated PV-11 virus (antigen) or with 1 ␮g/ml concanavalin-A (Sigma, St. Louis, MO, USA) and incubated in a humidified 5% CO2 incubator at 37 ◦ C. The cell supernatants were collected and the levels of IFN-␥, IL-4, IL-2 and IL-12 were measured using BD Pharmingen Opt EIA kit according to the manufacturer’s instructions. Briefly, 96-well microtiter plate was coated with capture antibody for IFN-␥, IL-4, IL-2 and IL-12 and incubated overnight at 4 ◦ C. The wells were aspirated, washed with PBS and incubated with the supernatants for 2 h at RT. The wells were again aspirated, washed and incubated with anti-mouse IgG HRP for 1 h at RT. The contents of each well were aspirated, washed, incubated with 100 ␮l substrate solution for 30 min in dark at RT, stop solution was added and O.D.450 was measured. The amount of cytokines was evaluated using linear regression equation obtained from the O.D.450 values of the standards.

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2.5. Protective efficacy against rabies virus CVS challenge The challenge studies were conducted in accordance with the National Institutes of Health (NIH) potency test for rabies vaccine (Meslin et al., 1996). In the experimental protocol, immunized mice were anesthetized and inoculated with 50 LD50 /30 ␮l of Challenge Virus Standard (CVS-11), strain by intracerebral route two weeks after the second booster. Subsequent to challenge, mice were observed for rabies-specific paralytic symptoms or death for 14 days, which were confirmed by Fluorescence Antibody test (FAT). Surviving mice were observed for another 2–3 weeks to ensure their well-being; after which they were euthanized to render them free of any pain or suffering. Survivorship rates obtained for different groups were assessed and Kaplan- Meier curves for survival were plotted. 3. Results 3.1. Supplementation of rabies DNA vaccine with alum elicited highest anti-glycoprotein antibody titer BALB/c mice immunized with Rabies DNA vaccine supplemented with 20% alum generated the highest IgG antibody titer starting with 1 IU/ml at 14th day post primary immunization, reaching up to 2 IU/ml at 28th day, which was maintained till 42nd day. On the other hand, supplementation of DNA vaccine with all other adjuvants, namely Immuvac, Montanide ISA 720 VG and Emulsigen-D led to slight increase in antibody titer from 0.5 to 1 IU/ml after two booster shots (Fig. 1a). Antibody isotyping was also conducted for all the groups and it was found that the DNA vaccine formulation with alum gave Th2 bent of immune response (Fig. 1b and c). The adjuvant Montanide ISA 720 VG also heightened humoral response but to a lesser extent than alum. Supplementation of vaccine with Mycobacterium W cells generated IgG titre with IgG1/IgG2a ratio <1, indicating cell-mediated immunity enhancement. However, supplementation of DNA vaccine with Emulsigen-D resulted in mixed Th1-Th2 response as reported in our prior study (Kaur et al., 2010). 3.2. Alum adjuvanted RDV induced highest RVNA titer RVNA titer was determined by RFFIT and it was found that RDV along with alum induced the highest titer of 4 IU/ml at 42nd day post primary immunization (Fig. 2). All the other three adjuvants elicited similar pattern of increase in RVNA titer from 1 IU/ml at 14th day to 2 IU/ml at the time of second booster, which was recorded to be constant till the time of challenge. The DNA vaccine construct alone led to comparatively low RVNA titer of 1 IU/ml, while the vector as well as healthy control generated negligible titers. 3.3. Cytokine estimation Upon in vitro stimulation of splenocytes obtained from immunized mice of different groups at 42nd day, the culture supernatants were harvested and cytokines were estimated. The vaccine formulation with Immuvac showed a heightened Th1 response with higher values of IFN- ␥ than that of IL-4, IL-2 and IL-12 (Tables 1 and 2). Supplementation with Montanide ISA 720 VG showed enhanced Th2 response, as also indicated by IgG1/IgG2a ratios. Addition of alum skewed the response towards Th2 arm while, Emulsigen-D addition illustrated mixed response with 780.62 ± 3.18 pg/ml IFN- ␥, 836.34 ± 5.40 IL-4, 45.27 ± 3.62 pg/ml IL-2 and 1590.48 ± 2.96 pg/ml IL-12 after 72 h of stimulation (Tables 1 and 2). RDV alone showed Th1 bent of immune

Fig. 1. Total IgG titer and IgG isotyping in mice immunized with rabies DNA vaccine supplemented with different adjuvants. Sera was collected and pooled group-wise from RDV immunized mice at 13th, 27th and 41st day post primary immunization. Total IgG titer was estimated using Reference sera test (A). IgG1 titer (B) and IgG2a titer (C) were measured using ELISA and represented as geometric mean of absorbance values (O.D.450 ) obtained from pooled serum samples (in triplicates) from each group.

response, while vector control and healthy control showed negligible cytokine induction. 3.4. Anti-Rabies virus protective efficacy of RDV with different adjuvants The immunization of RDV with different adjuvants was carried out according to NIH guidelines. After two weeks of second booster, the mice were challenged with 50 LD50 of CVS strain, and observed daily for rabies specific symptoms till 14 days and death of mice was recorded. As shown in Fig. 3, RDV supplemented with Alum as well as Emulsigen-D conferred highest protection (80%) to mice against the lethal rabies challenge. Immuvac and Montanide ISA 720 VG

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Table 1 IFN-␥ and IL-4 levels from splenocytes; 24 h, 48 h and 72 h post in vitro stimulation. Data are expressed as mean values ± S.D. of triplicates. Groups

Vaccine

I II III IV V VI VII

RDV + I RDV + M RDV + A RDV + E RDV Vector Control Healthy Control

IFN-␥

IL-4

24 h

48 h

72 h

24 h

48 h

72 h

680.14 ± 3.72 340.26 ± 2.84 385.35 ± 4.58 453.27 ± 3.62 270.35 ± 3.12 22.15 ± 1.02 12.56 ± 1.24

864.20 ± 4.02 480.32 ± 2.12 426.66 ± 5.60 649.55 ± 3.82 357.20 ± 3.44 23.57 ± 1.12 13.67 ± 1.88

1004.2 ± 5.16 560.40 ± 2.22 576.66 ± 4.12 780.62 ± 3.18 460.35 ± 3.80 23.65 ± 2.02 11.95 ± 1.16

349.30 ± 3.26 659.38 ± 3.74 688.42 ± 2.18 413.65 ± 4.66 93.64 ± 4.40 23.17 ± 2.44 11.42 ± 1.94

483.22 ± 3.84 890.64 ± 3.44 862.34 ± 2.20 630.50 ± 5.34 115.27 ± 4.88 22.82 ± 1.61 12.34 ± 1.20

590.48 ± 2.96 1004.20 ± 5.50 950.60 ± 2.80 836.34 ± 5.40 135.28 ± 4.60 22.64 ± 1.42 12.96 ± 1.08

Table 2 IL-2 and IL-12 levels from splenocytes; 24 h, 48 h and 72 h post in vitro stimulation. Data are expressed as mean values ± S.D. of triplicates. Groups

Vaccine

I II III IV V VI VII

RDV + I RDV + M RDV + A RDV + E RDV Vector Control Healthy Control

IL-2

IL-12

24 h

48 h

72 h

24 h

48 h

72 h

340.26 ± 2.84 40.15 ± 3.12 100 ± 1.02 78.62 ± 3.18 25.35 ± 3.21 22.15 ± 1.02 14.11 ± 0.56

120.32 ± 2.12 36 ± 0.24 52 ± 2.02 64.55 ± 3.82 15.20 ± 3.44 23.02 ± 1.04 18.45 ± 2.11

150.40 ± 2.22 32 ± 1.6 25 ± 3.62 45.27 ± 3.62 10.35 ± 3.80 19.04 ± 2.02 11.53 ± 3.1

1349.30 ± 3.26 688.42 ± 2.18 659.38 ± 3.74 1000 ± 2.4 349.30 ± 3.26 16.56 ± 2.11 12.21 ± 1.12

1483.22 ± 3.44 862.34 ± 2.20 890.64 ± 3.44 1200 ± 4.5 483.22 ± 3.84 17.82 ± 1.5 10.41 ± 0.85

2100 ± 6.3 950.60 ± 2.80 1004.20 ± 5.50 1590.48 ± 2.96 590.48 ± 2.96 18.45 ± 1.14 12.43 ± 1.1

Fig. 2. Rabies virus neutralization antibody (RVNA) titers determined by RFFIT. RVNA titers were determined from pooled sera samples collected from mice immunized with RDV supplemented with adjuvants at 13th, 27th and 41st day post primary immunization. RVNA titer equivalent to 0.5 IU/ml is the minimum adequate titer against rabies as recommended by WHO.

supplementation led to 70% survival post intracerebral viral challenge of immunized mice. Naked DNA vaccine immunization also imparted substantial protection (65%). The Kaplan-Meier estimate showing percentage survival of BALB/c mice till 14 days post lethal challenge has been depicted in Fig. 3. 4. Discussion The global framework for rabies control is largely dependent on ensuring affordable human vaccines and therapeutics, prompt post exposure treatment as well as mass dog vaccinations to tackle the disease at its source (WHO, 2016). Human infection mediated through canine rabies is of immense concern, as 59,000 human deaths occur every year and dog bites are responsible for approximately 95% of them. Therefore, control and elimination of canine rabies at the source is of huge public health implication. Timely and suitable post-exposure treatment is very expensive and largely unaffordable in Asian and African countries, where the majority of cases are inflicted. Under this scenario, the most cost-effective way to eliminate rabies is to prevent it at its animal source. Sustainable vaccination of 70% of the at-risk dog population is recognized as the key to eliminate the disease in endemic areas. Several studies conducted across the globe like Ethiopia (Coetzer et al., 2016),

Australia (Sparkes et al., 2016), Western Hemisphere especially Haiti (Schildecker et al., 2016), Middle East (Baghi et al., 2016) Japan (Kwan et al., 2016) have demonstrated the feasibility of mass vaccination. Furthermore, mass vaccination is more cost-effective than treating rabies exposures cases in humans. Under the canine rabies elimination program conducted at Philippines and supported from the Bill and Melinda Gates Foundation; it was observed that the mass dog vaccination campaigns carried out in each village were more cost-effective than fixed-site campaigns. Additionally, the average costs (in USD) per human life saved through PEP were $1620.28 in Cebu City and $1498 in Carmen. Costs per dog vaccinated ranged from $1.18 to $5.79 in Cebu City and $2.15 to $3.38 in Carmen (Miranda et al., 2015). The success of such mass vaccination programs with respect to cost effectiveness as well as rabies control is largely dependent on adequate resources, effective programme management, successful follow-up and evaluation. In this regard, the World Organization for Animal Health (OIE) and the World Health Organization (WHO), have formulated the five pillars of rabies elimination (STOP-R): socio-cultural, technical, organizational and political approaches (WHO, 2016). These key elements emphasize on promotion of responsible dog-ownership and dog population management practices, including dog vaccination; strengthening animal health and public health systems; ensuring sufficient supply of quality-assured canine rabies vaccines through vaccine banks; and promoting the One Health concept and intersectoral coordination through national and regional networks. Therefore, with these guidelines along with combined efforts of policy makers as well as international communities; dog-mediated human rabies elimination strategies could be a reality in near future. DNA vaccines have immense potential in this regard. Though, they have well established their efficacy and potency in several laboratory studies and clinical trials; their application in routine rabies prophylaxis has not yet been implemented (Ullas, 2012). Comparatively, higher immunizing DNA requisite along with poor immunogenicity in larger animals has restricted the largescale application. Tremendous efforts have been directed towards improvement of rabies DNA vaccination strategies. These largely relied on modification of glycoprotein gene, manipulation of vector backbone, improved vaccine formulations, advanced delivery technologies, and greater understanding of the immunological

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Fig. 3. Survival percentage of mice immunized with rabies DNA vaccine-adjuvant formulations. Immunized mice were challenged with 50LD50 CVS and observed for 14 days post challenge. Addition of alum to the vaccine culminated in 80% survival in mice as shown in the Kaplan-Meier curve, the highest amongst all other human approved adjuvants.

responses generated; targeting enhanced immunogenicity elicited and protection conferred. Of these, DNA vaccine formulations with potent adjuvants remain one of the most promising approaches for rabies DNA vaccination. Vaccine adjuvants include a whole range of compounds, like emulsions, micro and nanoparticles, liposomes, TLR agonists, PRR ligands and mineral salts which enhance the vaccine potential through diverse and sparsely characterized mechanisms of action (Fraser et al., 2007). Amongst the various adjuvants, alum has been largely used owing to its good safety profile and immune modulation with diverse antigens. However, some adverse reactions like muscle soreness, occasional fever, granuloma induction at the site of injection have been reported (Reed et al., 2009). More importantly, their inability to elicit cell-mediated immune responses has restricted their usage in several clinical manifestations which require containment and clearance of intracellular pathogens like in TB, malaria, leishmaniasis, leprosy and AIDS. This has encouraged alternative adjuvants, which would offer faster, robust and durable immunity with fewer adverse effects; alone or in combination for a variety of preclinical and clinical studies. Furthermore, greater interest and understanding of impact of adjuvants towards eliciting specific immune response has accelerated the research in this direction. Several classes of adjuvants like Alum derivatives, QS-21, MF59, MPL-based adjuvants, liposomes, cytokines and TLR agonists have been probed for specific modulation of immune system (Montomoli et al., 2011). Alum compounds (Ca3 (PO4 )3 , CTB, AFPL1) have been formulated for numerous vaccines, including tetanus, cholera, Hepatitis A virus, Hepatitis B virus, diphtheria, inactivated polio virus, Haemophilus influenzae type B, meningococcal vaccines (De Gregorio et al., 2008). Of these, AFPL1 has met with immense success and has been extensively used for humans, including children. Yet another adjuvant, QS-21, a Saponin has been used in a number of clinical trials targeting viral and parasitic diseases (herpes, influenza, HIV-1, malaria and hepatitis B) as well as cancer like breast, prostate and melanoma (Kensil and Kammer, 1998). Amongst emulsions, MF59 and AS03 have been licensed and used

in Europe for H1N1 pandemic flu campaign the 2009 (Clark et al., 2009). However, the US FDA has not yet licensed either of these adjuvants. Liposome based strategy using HBV surface antigen as VLP vaccine is also commercially available (Lobaina et al., 2005). Furthermore, HPV vaccines, Gardasil and Cervarix employing the VLP technology have been approved for clinical use (Harper et al., 2006; Tay et al., 2008). The ability of TLR agonists as therapeutics has also been investigated. Amongst, this class of novel adjuvants, monophosphoryl lipid A (MPL) is most widely used. MPL, alone or in combination (along with alum and QS-21) as well as in both liposome and emulsion formulations has been tested. Plasmodium falciparum recombinant antigen RTS,S in conjunction with MPL formulated with QS21 (AS02) confers 37% reduction of malaria infection (Alonso et al., 2004). On the other hand, AS04 (MPL and alum) is a constituent of licensed HBV vaccine, Fendrix (Beran, 2008). An ideal adjuvant depends on several factors including immunizing antigen, specific pathogen, animal species, route of immunization, and type of immunity needed and therefore, needs to be assessed for each and every vaccine formulation. In our previous study, we described generation and optimization of DNA vaccine formulation, which imparted complete protection in preand post-exposure efficacy analysis in murine model of rabies (Kaur et al., 2010, 2009). The pre-clinical safety of RDV has also been established by acute as well as repeated dose toxicity studies in Swiss Albino mice and Wistar rats respectively (Garg et al., 2014). The feeding pattern, organ weight as well as immunotoxicological parameters remained unperturbed even after 10X therapeutic dose administration in Wistar rats. The hematological as well as serological parameters did not exhibit any dose dependent variation. The vital organs also did not show any major alteration from normal in the experimental groups. Though, certain abscesses were observed at site of injection in vaccine supplemented with Emulsigen-D group, no such phenomenon was seen in RDV alone group, reinforcing the claim of safety of RDV (Garg et al., 2014). In addition to Emulsigen-D used in previous studies, here, we have analyzed the potential of Alum, Immuvac and Montanide ISA

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720 VG in adjuvanating rabies DNA vaccine, pgp.LAMP-1. It was observed that maximal enhancement of anti-glycoprotein antibody titer was observed upon supplementation with Alum. Furthermore, antibody isotype as well as cytokine profiling indicated that Alum skewed the immune response towards the protective Th2 type. The effect was either not prominent or mixed in case of other formulations. These combinations were also able to elicit rapid virus neutralizing antibody responses. Substantial increment in the RVNA titer was also observed in case of Alum usage. It is imperative to note that this titer is 8 times more than the minimum protective titer recommended by WHO. This elevation could be corroborated with the subsequent protective effect against the lethal rabies challenge. The immunization schedule as well as challenge study was conducted for assessing the protective efficacy of RDV with different adjuvant formulations, in accordance with the NIH guidelines (Meslin et al., 1996). It has been known that protection in the NIH potency test correlates well with serum virus neutralizing antibody (VNA) responses to rabies virus glycoprotein (Crick, 1974; Turner, 1974; Wunderli et al., 1991). Therefore, though, this test poses an extremely harsh challenge, that too synthetic, but is widely accepted by the scientific community as a stringent experimental test. Other methods like intranasal challenge are still in infantile stage and they don’t promise that the results from challenge would have lesser variation than seen with IC challenge (Lewis et al., 2013). The results demonstrate that RDV supplemented with Alum as well as Emulsigen-D conferred highest protection (80%) to mice against the lethal rabies challenge with 50 LD50 of CVS strain. CVS-11 is a well characterized strain, developed from the original Pasteur rabies virus (isolated in 1882) by adaptation in mice. It has been widely approved for vaccine production, recommended for use in RVNA tests as well as for in vivo challenge studies (Patel et al., 2015; Yu et al., 2013). Thus Alum is an effective adjuvant in context with rabies vaccination and comparable to Emulsigen-D, as determined in the previous study (Kaur et al., 2010). Furthermore, this study emphasizes on the RDV pgp.LAMP-1 − Alum formulation for effective rabies prophylaxis, particularly given that the Alum is already approved for human vaccine use. DNA vaccines have come a long way from their conceptualization till date. Despite, uniqueness of characteristics like in vivo antigen formation, processing, presentation and exploitation of host’s immunological machinery for disease prevention, they have often faced challenge from contradictory school of thoughts (Grunwald and Ulbert, 2015). Various clinical trials on several human subjects have revealed that there are no adverse effects like autoimmunity, transfer of resistance markers or integration of DNA vaccine into the genome (Sardesai and Weiner, 2011). Thus, DNA vaccine technology deserves serious consideration from the researchers world-wide, especially in case of rabies, where mass immunization drives are the only solution for reduction of disease related mortality. The potential of Rabies DNA Vaccine pgp.LAMP-1 – Alum formulation could be harnessed for this purpose.

Contributions R.G., M.K., R.P. and R.B. designed the study. R.G., M.K., A.S. performed the experiment and analyses. R.G., M.K. and R.B. wrote the manuscript. All authors have read and approved the final manuscript.

Funding This study was supported by a Grant from the Department of Science and Technology, Government of India.

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