Canine zona pellucida glycoprotein-3: Up-scaled production, immunization strategy and its outcome on fertility

Vaccine 33 (2015) 133–140

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Canine zona pellucida glycoprotein-3: Up-scaled production, immunization strategy and its outcome on fertility Abhinav Shrestha a , Sudeepa Srichandan b , Vidisha Minhas a , Amulya Kumar Panda b , Satish Kumar Gupta a,∗ a b

Reproductive Cell Biology Laboratory, National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi 110 067, India Product Development Cell, National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi 110 067, India

a r t i c l e

i n f o

Article history: Received 22 July 2014 Received in revised form 20 September 2014 Accepted 2 November 2014 Available online 13 November 2014 Keywords: Adjuvant CpG motif Immunocontraception Recombinant protein produced in fermentor Zona pellucida glycoprotein-3

a b s t r a c t Zona pellucida (ZP) glycoproteins based contraceptive vaccines have been proposed for the management of wildlife population. In the present study, a fusion protein encompassing promiscuous T cell epitope of tetanus toxoid [TT; amino acid (aa) residues 830–844] followed by a dilysine linker and an ectodomain of dog ZP3 (ZP3; aa residues 23–348) without any affinity tag (TT-KK-ZP3) has been expressed in Escherichia coli. The recombinant protein was successfully produced in fed-batch fermentor and purified. The average yield of purified refolded protein was 12.20 ± 0.61 mg/2 g wet cell pellet. Female FvB/J mice immunized with the varying doses of recombinant TT-KK-ZP3 supplemented with alum/PetGel A as adjuvants following a three injection schedule, showed dose dependent increase in serum IgG titer. Antibodies against TT-KK-ZP3 recognized native mouse/dog ZP and significantly inhibited mouse in-vitro fertilization (p = 0.012). Immunized mice showed significant reduction in fertility (p < 0.05). Higher antibody titers were associated with a decrease in the number of pups born to the immunized female mice. To reduce the number of injections, two injection schedule using various dose combinations of TT-KKZP3 supplemented with alum revealed lower immunogenicity and contraceptive efficacy as compared to the three injection schedule. To overcome this, CpG motif was included in addition to alum and both intraperitoneal and intranasal route of immunization following the two injection schedule was investigated. Inclusion of CpG significantly enhanced the antibody titer and improved contraceptive efficacy. In the mice immunized following intraperitoneal route, serum/vaginal IgG and in the mice immunized through intranasal route, vaginal IgA seemed to be important for curtailment in fertility. To conclude, the recombinant protein described herein may be a good candidate for developing contraceptive vaccine for the wildlife population management, in particular street dogs. © 2014 Elsevier Ltd. All rights reserved.

1. Introduction Contraceptive vaccines based on zona pellucida (ZP) glycoproteins have been used to manage population of feral horses (Equus caballus) at Assateague Island National Seashore, MD, USA and white-tailed deer (Odocoileus virginianus) inhabiting Fire Island National Seashore, NY, USA without any significant debilitating short or long-term health effects in the vaccinated animals [1–4]. In Australia and New Zealand, extensive studies have been done to explore the potential of ZP-based contraceptive vaccine for controlling the population of eastern grey kangaroos (Macropus giganteus) and brushtail possum (Trichosurus vulpecula) [5,6]. Besides

∗ Corresponding author. Tel.: +91 11 26741249; fax: +91 11 26742125. E-mail address: [email protected] (S.K. Gupta). http://dx.doi.org/10.1016/j.vaccine.2014.11.003 0264-410X/© 2014 Elsevier Ltd. All rights reserved.

controlling habitation conflict between humans and wild animals, management of wildlife population by the use of contraceptive vaccines would have an important bearing on the spread of zoonotic diseases. Globally, zoonoses have a significant role in various infectious diseases in humans [[7,8], http://www.cdc.gov/ncezid/]. For example, dogs are one of the main reservoirs and carriers of rabies virus. To control the population of street dogs, currently either spaying of female dogs or castration of male dogs is carried out. These measures have failed to effectively control their population, resulting in the increase of rabies infection in several developing countries [9,10]. In this regard, contraceptive vaccines may provide an exciting option for the management of street dog population. Such vaccines can be used in animals on the basis of “herd immunization” approach, in which, immunization outcome need not to be evaluated on the individual basis, but an overall decline in the population of target species is achieved.

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Immunization of bitches with native porcine ZP led to the inhibition of fertility, which was associated with altered ovarian functions [11]. However, there has been concern on the quality of native porcine ZP employed in these investigations and hence candidature of porcine ZP-based contraceptive vaccine to control fertility in dogs is debatable. Use of recombinant zona proteins may be a viable proposition to develop ZP-based contraceptive vaccine to circumvent the problems associated with the limited availability of native ZP, tedious process of their purification and probability of contamination by other ovary associated proteins [12]. The feasibility of generating infertility in female dogs immunized with recombinant dog ZP glycoprotein-3 (ZP3) conjugated with diphtheria toxoid (DT) has been previously demonstrated [13]. However, to avoid chemical conjugation of recombinant dog ZP3 with a carrier protein (DT), as it might result in batch to batch variation in vaccine preparation, and to produce recombinant protein without any affinity tag (to avoid any untoward side effect subsequent to immunization due to presence of the tag [14]), a tag-free fusion protein encompassing promiscuous T cell epitope of tetanus toxoid [TT; amino acid (aa) residues 830–844] followed by a dilysine linker and an ectodomain of dog ZP3 (aa residues 23–348) was expressed (TT-KK-ZP3) [15]. In order to produce TT-KK-ZP3 in larger amount to undertake further studies, purified protein from transformed M15 (pREP4) Escherichia coli cells grown and induced in fed-batch fermentor, revealed a major band of ∼30 kDa instead of expected 39 kDa on SDS-PAGE (Unpublished observations). Hence in the present study, to obtain full length TT-KK-ZP3, we report modified expression strategy, wherein both the expression vector and host cells were changed and process to grow transformed E. coli in fed-batch fermentor was optimized. Before initiating the contraceptive efficacy studies in beagle/non-descript female dogs, we report in this manuscript the immunization strategy to obtain optimum contraceptive efficacy of the purified recombinant TT-KK-ZP3 in female mice. Blast analysis of mouse ZP3 (GI no. 1663714) with dog ZP3 (GI no. 50979000) sequence obtained from National Center for Biotechnology Information (NCBI), USA database revealed 66% amino acid sequence identity and antibodies against recombinant dog ZP3 cross-react with mouse ZP [15].

2. Materials and methods 2.1. Cloning and expression of recombinant TT-KK-ZP3 A codon optimized synthetic gene was designed to encode recombinant protein encompassing promiscuous T cell epitope of TT (aa residues 830–844) followed by dilysine linker (KK) and an ectodomain of dog ZP3 (23–348 aa residues), which was obtained from GenScript Corporation (Piscataway, NJ, USA) in pUC57 vector (TT-KK-ZP3). Synthetic gene was flanked by Nde1 and BamH1 restriction sites at 5 and 3 ends, respectively. A stop codon (TAA) was present upstream of BamH1 site to avoid concomitant expression of His6 -tag. Using standard protocols, gene was cloned in pET-22b(+) expression vector and BL21[DE3]pLysS E. coli cells (Stratagene, CA, USA) were transformed with the plasmid encoding TT-KK-ZP3. Transformed cells were grown at 37 ◦ C until the OD600 reached between 0.4 and 0.6 and the expression of recombinant protein was induced with optimized concentration of 1.0 mM isopropyl-␤-d-thiogalactopyranoside (IPTG; SigmaAldrich, St. Louis, USA) for 3 h. Expression of recombinant protein was checked by 0.1% SDS-10% PAGE and confirmed by Western blot using monoclonal antibody MA-451 (specific for ZP3 from various species) [16] essentially as described previously [13].

2.2. Production of recombinant TT-KK-ZP3 in fed-batch fermentor Expression of the recombinant protein at large scale was carried out using fed-batch fermentor in 5 l working volume [17]. Briefly, cells were grown in batch mode for 3 h and nutrient feeding was initiated to obtain high cell density. Concentration of yeast extract and glucose in concentrated feed was 250 g/l each. Feed rate was 200 ml/h for the last 3 h before induction with IPTG followed by 100 ml/h for next 3 h post-induction. Expression of the recombinant protein was induced by the addition of 1.0 mM IPTG for 4 h when OD600 reached approximately 25.0. 2.3. Purification of the recombinant TT-KK-ZP3 Recombinant protein was expressed in the form of inclusion bodies (IBs). For purification of IBs, 2 g wet pellet of the bacterial cells was resuspended in 20 ml of 50 mM Tris buffer, 5 mM EDTA; pH 8. Cells kept in ice were sonicated using Branson Sonifier-450 (Branson Ultrasonic Corp., CT, USA) pulse for 10 min (3 s on, 2 s off) at 40% output. Sonicated cells were centrifuged at 8000 × g for 30 min at 4 ◦ C and supernatant discarded. The pellet was resuspended in 20 ml of wash buffer [50 mM Tris, pH 8, supplemented with 2% sodium deoxycholate (Amresco, Solon, Ohio, USA), 100 mM NaCl and 5 mM EDTA], incubated for 20 min at room temperature (RT) followed by sonication for 5 min. Washing step was repeated one more time without incubation. Subsequently, IBs pellet was washed with 20 ml of 50 mM Tris; pH 8 and finally washed with double distilled water. Pellet represents purified IBs. The process for solubilization and refolding of recombinant TT-KK-ZP3 was same as described previously [18]. Refolded protein was filtered, concentrated and quantified using BCA Protein Estimation Kit (Pierce, Rockford, IL, USA). 2.4. Immunization studies Inbred FvB/J mice, 8–10 weeks of age, kept under the conventional containment levels at the Small Experimental Animal Facility, National Institute of Immunology, New Delhi were used. These studies were conducted as per the guidelines of Institutional Animals Ethics Committee (IAEC). For three injection schedule, female mice (n = 10 per group) were immunized subcutaneously with 25, 50 and 125 ␮g of recombinant TT-KK-ZP3 with either alum (Sigma) (200 ␮g aluminum content/injection/mouse) or 5% MontanideTM PetGel A (Seppic, Paris, France) [19] as adjuvants along with the appropriate adjuvant controls. Two boosters were given on days 21 and 42 intraperitoneally with the same amount of recombinant proteins and adjuvants. Sera were collected through retro-orbital bleeding on days 0, 35 and 56. For two injection schedule, female mice (n = 10 per group) were primed with 125 ␮g TT-KK-ZP3 and boosted with 25 ␮g of protein or primed with 50 ␮g TT-KK-ZP3 and boosted with either 25 ␮g or 50 ␮g of protein. All injections were formulated with alum only. First injection was given subcutaneously and booster was given on 28th day intraperitoneally. Sera were collected through retroorbital bleeding on days 0, 21 and 42. Keeping in view the feasibility of priming and boosting with same dose and route during field application, 50 ␮g + 50 ␮g recombinant TT-KK-ZP3 was chosen for optimization of route of immunization and simultaneously evaluating the effect of including CpG motif (ODN-2006, class-B, Hycult Biotech, Uden, The Netherlands) on the immunogenicity and contraceptive efficacy in the following groups of mice (n = 10 per group): (i) TT-KK-ZP3 + alum + 50 ␮g of CpG motif; through intraperitoneal route.

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Fig. 1. Kinetics of bacterial biomass growth, SDS-PAGE and immunoblot profile of recombinant TT-KK-ZP3. Panel A shows kinetics of bacterial biomass growth and recombinant protein production in fed-batch fermentation of E. coli expressing recombinant TT-KK-ZP3. Fermentation was done in 5 L batch at 37 ◦ C and pH 7. Arrow indicates point of induction of bacterial cells with 1 mM IPTG. Panel B shows Coomassie stained SDS-PAGE analysis of fermentor samples at different time intervals. Lanes 1, 2, 3 represent uninduced cells at 4, 5, 6 h of fed-batch growth and lanes 4, 5, 6, 7 represent whole cell lysate of the cells induced with 1 mM IPTG for 1, 2, 3, 4 h, respectively. Panel C represents Coomassie stained SDS-PAGE profile of purified TT-KK-ZP3 from inclusion bodies from three different batches as shown in lanes 1, 2 and 3. Panel D shows Western blot profile of the purified TT-KK-ZP3 from fermentor; Lane M represents molecular weight markers.

(ii) TT-KK-ZP3 + alum + 50 ␮g of CpG motif; through intranasal route. (iii) Adjuvant control group. Priming and boosting were done with same route and formulation within each group. Booster was given on 28th day after first injection and sera were collected through retro-orbital bleeding on days 0, 21 and 42. In addition, the vaginal washes were also collected on day 42 by flushing 50 ␮l of the sterile 50 mM PBS, pH 7.4 in the vaginal lumen.

2.7. Statistical analysis The statistical significance of antibody titers of pregnant and non-pregnant mice within each group was performed by using paired student’s t-test assuming unequal variance. Similar method was used to assess significance of the contraceptive efficacy achieved in various groups as compared to the control. A p-value of <0.05 was considered to be statistically significant. The Pearson correlation coefficient (r-value) between antibody titers and number of pups born to the immunized mice was calculated using linear fit by Origin50 software.

2.5. ELISA 3. Results The serum IgG titer against recombinant TT-KK-ZP3 was determined essentially as described previously [18]. Antibody titers are expressed as antibody unit (AU) which represents inverse of serum dilution showing an OD490 of 1. For measuring IgA and IgG titers in vaginal washes, vaginal washes of pregnant and non-pregnant mice were pooled, diluted 1:1 with PBS, and processed essentially as described above, except that HRPO (horseradish peroxidise) conjugated goat anti-mouse IgA secondary antibody (Pierce) was used for determining IgA titer. Vaginal IgG/IgA titer is expressed as absorbance at 490 nm (OD490 ).

2.6. In vivo contraceptive efficacy studies Two immunized female mice were co-habitated with one male mouse for 15 days after last bleed (days 56 or 42 in case of 3 or 2 injections schedule, respectively). Male mice were rotated every 5th day during mating study. Number of pups born per pregnant female mouse was counted.

3.1. Fed-batch fermentor yielded high amount of refolded recombinant TT-KK-ZP3 with secondary structure The schematic diagram of the construct used to express recombinant TT-KK-ZP3 in E. coli is shown in Supplementary Fig. 1. Transformed BL21[DE3]pLysS E. coli cells growing in fed-batch fermentor when induced with 1 mM IPTG at an OD600 of ∼25.0, showed time dependent increase in the expression of recombinant protein (Fig. 1A). SDS-PAGE analysis of the cells harvested at different time points before and after the addition of IPTG showed a prominent band at ∼39 kDa only in the induced cells, which increased in size as a function of time (Fig. 1B). Recombinant TTKK-ZP3 was purified from 2 g wet cell pellet as described in Section 2. The SDS-PAGE profile of 3 different lots of the purified protein is shown in Fig. 1C. Western blot analysis of the purified refolded protein revealed a band of ∼39 kDa (Fig. 1D). The average yield for the purified TT-KK-ZP3 from 3 lots was 12.20 ± 0.61 mg/2 g wet cell pellet (data not shown).

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(C) Fig. 2. Serum IgG titer and fertility status of mice immunized with recombinant TT-KK-ZP3 following three injection schedule: Panels A and B show serum IgG titers of female mice (n = 10) immunized with 25, 50 and 125 ␮g of TT-KK-ZP3 supplemented with either alum or PetGel A as adjuvants on days 0, 35 and 56. Antibody titers are expressed as AU and represent the mean ± S.E.M of the group of mice immunized with respective dose of recombinant protein. Panel C shows fertility status of the immunized mice in the respective group and number of pups born (mean ± S.E.M) per female.

Fluorescent spectroscopy and CD spectrum of the purified refolded TT-KK-ZP3 performed as described previously [18], revealed the presence of defined secondary structure (Supplementary Fig. 2). 3.2. Recombinant TT-KK-ZP3 is immunogenic in female mice and curtails fertility Female mice immunized with 25, 50 and 125 ␮g of recombinant TT-KK-ZP3 per injection supplemented with either alum (Fig. 2A) or PetGel A (Fig. 2B) following a three injection schedule showed high serum IgG titer. Antibody titers increased with an increase in the amount of the antigen in presence of either of the adjuvants. Boosting effect was observed after every injection. Anti-TT-KK-ZP3 antibodies recognized native mouse and dog ZP matrix (Supplementary Fig. 3) and also inhibited mouse in vitro fertilization significantly (p = 0.012) as compared to pre-immune control serum (Supplementary Table 1). Mating studies of the group of mice immunized with varying doses of recombinant TT-KK-ZP3 either with alum or PetGel A showed failure of conception ranging from 50 to 90% (Fig. 2C). In the group of mice immunized with 125 ␮g TT-KK-ZP3 per injection with alum, the number of pups born was 0.6 ± 0.60 (mean ± S.E.M) per female which is significantly less (p = 9.29 × 10−8 ) as compared to 8.4 ± 0.76 in adjuvant control (Fig. 2C). Interestingly, mice that failed to conceive subsequent to immunization, showed significantly higher serum IgG titers against TT-KK-ZP3 (p < 0.05) as compared to those who became pregnant

even after immunization (Fig. 3). The Pearson correlation coefficient (r-value) analyses revealed that higher the antibody titers against TT-KK-ZP3, lower is the number of pups born to the immunized animals (Fig. 2C). Further, with an attempt to reduce the number of injections, mice were immunized following two injection schedule as described in Section 2. In this experiment, highest antibody titers against TT-KK-ZP3 (32.2 ± 3.45 × 103 AU) were observed in the group of mice immunized with 125 + 25 ␮g protein and it showed 40% infertility (Table 1). Overall antibody titers and contraceptive efficacy achieved following 2 injection schedule was lower as compared to 3 injection schedule (Fig. 2, Table 1). 3.3. Incorporation of CpG motif led to an increase in the immunogenicity and contraceptive efficacy In order to enhance the efficacy of two injection schedule, in the next step, CpG motif was included as a co-stimulant along with alum and dose of the recombinant protein was kept at 50 + 50 ␮g as described in Section 2. Separate groups of mice were immunized intraperitoneally as well as intranasally, with an aim to simultaneously investigate that out of systemic (mediated by intraperitoneal route) and mucosal (mediated by intranasal route) immune response, which one is more effective in curtailing fertility. Mice immunized intraperitoneally with TT-KK-ZP3 supplemented with alum and CpG motif showed significantly higher serum IgG titer as compared to the mice immunized with any of the three

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Fig. 3. Correlation between serum IgG titer and fertility status of mice immunized with recombinant TT-KK-ZP3 following three injection schedule: Panels A and B show scatter diagram of anti-TT-KK-ZP3 antibody titers of day 56 bleed. Antibody titers of pregnant and non-pregnant mice are represented by different symbols in group of mice immunized with 25, 50 and 125 ␮g of TT-KK-ZP3 supplemented with alum (Panel A) and PetGel A (Panel B), respectively. The values in parenthesis represent p-value of the difference in the serum anti-TT-KK-ZP3 antibody titers between pregnant and non-pregnant mice in the respective group of immunized animals.

doses of TT-KK-ZP3 + alum from the previous two injection schedule experiment (p-value: 0.034, 0.0108 and 0.005 for 125 + 25, 50 + 50 and 50 + 25 ␮g groups, respectively) (Tables 1 and 2). Group of mice immunized with TT-KK-ZP3 supplemented with alum and CpG motif following intraperitoneal route, showed mean serum IgG titer of 53.9 ± 8.82 (A.U × 103 ) at day 42 of immunization and 50% immunized females failed to conceive (Table 2). On the other hand, animals immunized without CpG motif but with same amount of TT-KK-ZP3 + alum from the previous two injection schedule experiment following subcutaneous and intraperitoneal route (as described in Section 2), had shown mean serum IgG titer of 26.5 ± 2.28 (A.U × 103 ) at day 42 of immunization and reduction of fertility by 30% which were lower as compared to group

of mice where CpG motif was included as an additional stimulant (Tables 1 and 2). 3.4. Relevance of serum IgG and vaginal IgG/IgA antibodies generated by recombinant TT-KK-ZP3 in inducing infertility As mentioned in previous section, to study the relative relevance of systemic versus mucosal immune response for achieving infertility, separate groups of female mice were immunized with recombinant TT-KK-ZP3 supplemented with alum and CpG motif by intraperitoneal as well as intranasal routes following two injection schedule (50 + 50 ␮g/injection) as described in Section 2. Serum and vaginal IgG titer against TT-KK-ZP3 were higher in intraperitoneal

Table 1 Serum IgG titer and fertility status of mice immunized with recombinant TT-KK-ZP3 protein supplemented with alum (two injection schedule). Immunogen

Mean serum IgG titer on day 42 (AU × 103 )

% Of animals failed to conceive

Pups/mated female (mean ± S.E.M.)

Alum TT-KK-ZP3 (125 ␮g + 25 ␮g) TT-KK-ZP3 (50 ␮g + 50 ␮g) TT-KK-ZP3 (50 ␮g + 25 ␮g)

<0.05 32.2 ± 3.45 26.5 ± 2.28 23.2 ± 2.53

0 40 30 30

9.1 4.7 5.3 6.2

± ± ± ±

0.65 1.49 1.19 5.71

p-Value of the number of pups born from immunized group as compared to control

p-Value of the serum IgG titers of pregnant and non-pregnant mice on day 42

N/A 0.018 0.015 0.11

N/A 3.77 × 10−6 5.86 × 10−7 5.88 × 10−8

Table 2 Antibody titer and fertility status of female mice immunized with recombinant TT-KK-ZP3 protein supplemented with alum and CpG motif (two injection schedule). Immunogen

Mean serum IgG titer on day 42 (AU × 103 )a , b

Mean vaginal IgG titer on day 42a , c

Mean vaginal IgA titer on day 42a , c

% of animals that failed to conceive

Pups/mated female (mean ± S.E.M.)

p-Value of the number of pups born from immunized group as compared to control

Adjuvant control TT-KK-ZP3 (50 ␮g + 50 ␮g) + alum + CpG (i.p.) TT-KK-ZP3 (50 ␮g + 50 ␮g) + alum + CpG (i.n.)

<0.05 53.90 ± 8.82

<0.05 1.61 ± 0.52

<0.05 0.58 ± 0.07

0 50

8.33 ± 0.55 3.00 ± 1.13

N/A 7.90 × 10−4

12.85 ± 1.22

0.50 ± 0.11

1.72 ± 0.64

50

3.60 ± 1.32

5.48 × 10−3

a b c

Serum IgG and vaginal IgG and IgA titers represent mean titer of all mice (pregnant and non-pregnant) within the group. Serum IgG titer is expressed as AU which represents inverse of serum dilution giving value of 1.0 at OD490 . Vaginal IgG/IgA titer is represented by absorbance at 490 nm (OD490 ) of vaginal wash (1:1 diluted with PBS).

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Fig. 4. Serum/vaginal IgG/IgA titer of pregnant/non-pregnant mice immunized with recombinant TT-KK-ZP3 supplemented with alum and CpG motif following two injection schedule: Panels A & B show serum IgG and vaginal IgG and IgA titer for pregnant and non-pregnant mice immunized through intraperitoneal route with 50 ␮g of recombinant TT-KK-ZP3 supplemented with alum and CpG motif following two injection schedule as described in Section 2. Panels C and D show serum IgG and vaginal IgG and IgA titer for pregnant and non-pregnant mice immunized through intranasal route in the same experiment.

group as compared to intranasal group (Table 2). In intraperitoneal group, serum (p = 1.57 × 10−5 ) as well as vaginal (p = 0.03) IgG titers were significantly higher in the mice that failed to conceive versus those who became pregnant (Fig. 4A and B). No significant difference in serum (p = 0.85)/vaginal (p = 0.92) IgG titers was observed in intranasal group between the pregnant and the non-pregnant mice (Fig. 4C and D). Interestingly, in the intranasal group, vaginal IgA (p = 0.01) titers were significantly higher in the group of mice that failed to conceive as compared to those who became pregnant (Fig. 4D).

4. Discussion Previously, we have reported expression of tag-free recombinant TT-KK-ZP3 using pQE-60 expression vector in M15 (pREP4) cells of E. coli [15]. Due to failure in up-scaling the production of recombinant protein at fermentor level, expression vector and host cell were changed, which led to successful production of full length protein (∼39 kDa) in fermentor with high yield and defined secondary structure. Immunization studies with recombinant protein in female mice following three injection schedule suggested a correlation between antibody titers against recombinant TT-KK-ZP3 and failure to conceive as well as the number of pups born among all the six groups of immunized animals. Alum was found to elicit more uniform immune response and resulted in better contraceptive efficacy as compared to PetGel A.

Achieving antibody mediated contraception is a phenomenon dependant on female genital tract (FGT) immunity which can be achieved either by eliciting mucosal immune response or by generating systemic antibody response, so that antibodies in the later case percolate into female genital tract [20]. Indeed, it has been shown that systemically administered monoclonal antibodies against Simian Immunodeficiency Virus (SIV) percolated into FGT of rhesus macaques and conferred protection from intra-vaginal challenge with SIV [21,22]. Though lacking in organized mucosa associated lymphoid tissue system (MALT), mice FGT is conventionally considered as a part of common mucosal immune system (CMIS) [23]. Presence of IgA secreting plasma cells in uterine tissues [24,25], ability of B cells to reach at FGT from other mucosal sites [26] and presence of secretary component in uterine epithelium, enable local production as well as transport of immunoglobulins; predominantly IgA in mice FGT upon activation of CMIS. Intra-nasal site which contains organized MALT, have been shown to be preferred route for elicitation of FGT immune response as compared to many other mucosal sites including intra-vaginal route [23,27,28]. Therefore in the current study, we wanted to investigate whether it is locally produced mucosal immune response or systemic immune response, which is more efficient in curtailing the fertility and that is why intraperitoneal and intranasal routes of immunization were evaluated simultaneously in final leg of the experiments. In the same experiment, in order to improve the immunogenicity and efficacy of two injection schedule, CpG motif was included

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as a co-stimulant with alum and simultaneously intraperitoneal and intranasal routes were investigated. Acting as TLR-9 agonist, CpG motifs are unmethylated small stretch of nucleotides of bacterial origin, which show synergism with alum as adjuvant and help to elicit higher immune response [29–33]. CpG motif has also been shown to be relevant for eliciting systemic as well as mucosal immune response [34,35]. Inclusion of CpG motif along with alum significantly improved the serum antibody titer (IgG) and efficacy of the vaccine as compared to only alum based formulation in a two injection schedule following intraperitoneal route. Also, CpG motif + alum based formulation could elicit effective mucosal immune response against recombinant TT-KK-ZP3 through intranasal route, whose outcome in terms of fertility, was comparable to intraperitoneal route. In this manuscript, we report for the first time the relevance of systemic (intraperitoneal) versus mucosal (intranasal) immune response in achieving contraception mediated by recombinant dog ZP3 supplemented with alum and CpG motif. Though, contraceptive efficacy achieved through intraperitoneal and intranasal routes was similar in terms of number of mice that failed to conceive, but considerable difference in serum/vaginal IgG/IgA titers were observed between the two groups. In intraperitoneal group there is significantly higher serum IgG titer as compared to intranasal group (p = 2.18 × 10−4 ) which can be attributed to the presence of high concentration of antigen presenting cells at the intraperitoneal site that would facilitate efficient uptake of the antigen, thus generating a robust systemic immune response [36]. High serum IgG titer in intraperitoneal group correlated with high vaginal IgG titer, which might be a result of passive transudation of serum IgG into vaginal lumen [37]. In this group, non-pregnant mice showed significantly higher serum/vaginal IgG as compared to that of pregnant mice. However, vaginal IgA was very less and it did not show any correlation with fertility status of mice. On the other hand, in case of intranasally immunized group, pregnant and non-pregnant mice showed no significant difference in the level of serum/vaginal IgG, but, there is a correlation between vaginal IgA titer and curtailment in fertility, as non-pregnant mice had significantly (p = 0.01) higher level of vaginal IgA as compared to pregnant mice. Although serum IgA was detectable in ELISA up to 1:100 dilution of serum, yet it was very low as compared to serum IgG present in intraperitoneal as well as intranasal group. We have not shown serum IgA titer, because in any of the groups the titer was very low and did not show any correlation with fertility status of mice and did not seem to be relevant for contraception. Even in intranasally immunized group, where we observed good mucosal immune response and infertile mice showed significantly higher vaginal IgA titer as compared to pregnant mice, serum IgA did not seem to correlate with either fertility status of mice or vaginal IgA titer. Further, low ratio of specific serum/vaginal IgA titer (as compared to very high serum/vaginal IgG ratio) and lack of correlation between their titers within individual mouse of the same group, suggest that vaginal IgA is not the result of passive transudation of serum IgA and hence might be locally produced secretory IgA. In a nutshell, this study suggests that by changing expression vector and host cell, production of recombinant TT-KK-ZP3 can be up-scaled at fermentor level with good yield. Also, despite of reducing the number of injections, immunogenicity and efficacy of recombinant TT-KK-ZP3 based contraceptive vaccine can be maintained or improved by employing combination of adjuvants. For achieving antibody mediated contraception, bio-availability of antibodies at effector site i.e. female genital tract is pertinent.

Conflict of interest statement The authors have declared that no competing interests exist.

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