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Tetanus vaccination with a dissolving microneedle patch confers protective immune responses in pregnancy
Journal of Controlled Release 236 (2016) 47–56
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Journal of Controlled Release journal homepage: www.elsevier.com/locate/jconrel
Tetanus vaccination with a dissolving microneedle patch confers protective immune responses in pregnancy E. Stein Esser a, AndreyA. Romanyuk b, Elena V. Vassilieva a, Joshy Jacob a, Mark R. Prausnitz b, Richard W. Compans a, Ioanna Skountzou a,⁎ a b
Department of Microbiology and Immunology and Emory Vaccine Center, Emory University School of Medicine, Atlanta 30322, Georgia School of Chemical and Biomolecular Engineering, Georgia Institute of Technology, Atlanta 30332, Georgia
a r t i c l e
i n f o
Article history: Received 3 December 2015 Received in revised form 24 April 2016 Accepted 16 June 2016 Available online 18 June 2016 Keywords: Dissolving microneedle patches Skin immunization Tetanus toxoid Mouse model Pregnancy Survival
a b s t r a c t Maternal and neonatal tetanus claim tens of thousands lives every year in developing countries, but could be prevented by hygienic practices and improved immunization of pregnant women. This study tested the hypothesis that skin vaccination can overcome the immunologically transformed state of pregnancy and enhance protective immunity to tetanus in mothers and their newborns. To achieve this goal, we developed microneedle patches (MNPs) that efficiently delivered unadjuvanted tetanus toxoid to skin of pregnant mice and demonstrated that this route induced superior immune responses in female mice conferring 100% survival to tetanus toxin challenge when compared to intramuscular vaccination. Mice born to MNP-vaccinated mothers showed detectable tetanus–specific IgG antibodies up to 12 weeks of age and complete protection to tetanus toxin challenge up at 6 weeks of age. In contrast, none of the 6-week old mice born to intramuscularly vaccinated mothers survived challenge. Although pregnant mice vaccinated with unadjuvanted tetanus toxoid had 30% lower IgG and IgG1 titers than mice vaccinated intramuscularly with Alum®-adjuvanted tetanus toxoid vaccine, IgG2a titers and antibody affinity maturation were similar between these groups. We conclude that skin immunization with MNPs containing unadjuvanted tetanus toxoid can confer potent protective efficacy to mothers and their offspring using a delivery method well suited for expanding vaccination coverage in developing countries. © 2016 Elsevier B.V. All rights reserved.
1. Introduction Tetanus is an acute disease caused by a neurotoxin produced by the anaerobic bacterium Clostridium tetani with motor and autonomous nervous system manifestations such as muscle rigidity and convulsive spasms [1]. Despite a 93% reduction of disease prevalence since launching of the Maternal and Neonatal Tetanus Elimination Initiative in 1988, tetanus has yet to be eliminated in 22 countries. In the areas where access to antenatal care is well below 50%, the incidence of neonatal tetanus is around 10% presenting a major public health concern [2–6]. In 2013 alone, material and neonatal tetanus claimed lives of several thousands of women and 50,000 newborns [7]. Once the disease is contracted, the fatality rate can be as high as 100% without hospital care and varies from 10% to 60% even with hospital care [8]. Disease incidence can be reduced through hygienic delivery practices and vaccination with Tdap (adult tetanus, diphtheria, pertussis vaccines). Vaccination with Tdap not only confers protective immunity to the mother, but also provides a passive immunity to the infants prior ⁎ Corresponding author. E-mail address: [email protected] (I. Skountzou).
http://dx.doi.org/10.1016/j.jconrel.2016.06.026 0168-3659/© 2016 Elsevier B.V. All rights reserved.
to the development of their own immune system [9,10]. Irrespective of the patient's prior immunization history, the Tdap vaccine should be administered at any time during pregnancy or immediately postpartum. However, in order to maximize passive antibody transfer, it is recommended that the Tdap vaccine be administered during the last trimester of pregnancy [11]. The current route for tetanus immunization is intramuscular delivery with syringes and needles. This approach presents several immunological and logistical disadvantages. With regards to immunological disadvantages, tetanus toxoid (TT) is a weak antigen [12], hence the addition of an adjuvant (Alum®) as well as repeated boosters are necessary to induce robust antibody titers and affinity maturation [13,14]. Another important problem for effective immunization is the immunotolerant state which results from pregnancy. Unless women have been immunized prior to pregnancy, the recommended immunization does not serve as a booster and only elicits a modest primary response. As for logistical problems to be addressed, particularly in rural areas of developing countries, those include requirement for cold chain, a critical bottleneck for vaccine storage and transportation; distribution and administration by trained personnel. Unfortunately the costs for their implementation are a limiting factor for effective
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administration and vaccination coverage. Thus, in rural areas it is difficult to complete the vaccination regimen; hence one or two tetanus vaccine doses as recommended by WHO are not sufficient to elicit effective responses in a population without pre-existing immunity [15,16]. Therefore new vaccination and administration approaches are urgently needed. Skin is recently gaining more attention as a vaccine target organ due to its immunological properties, which include an increased number of professional antigen presenting cells (APCs) compared to the muscle [17–19]. Antigen uptake initiates a cascade of innate immune responses resulting in its efficient presentation to naïve T and B cells that reside in draining lymph nodes proximal to the insertion site. Effective cross-talk between innate and adaptive immune responses generates more robust, longer-lasting immune protection [20,21]. We and other investigators have previously reported that skin immunization with metal or dissolving microneedle patches (MNPs) carrying various vaccines (influenza, inactivated poliovirus (IPV), measles, tetanus, HPV, rotavirus, BCG, malaria and RSV) confers immune responses at least non-inferior to conventional immunization as shown by lethal challenge studies, longevity of immune responses and breadth of immunity in a number of small-animal models [21–30]. In this study, we determined the immunological advantages of skin tetanus vaccination in pregnancy using MNPs or intradermal injection (ID) in mice and compared the results to intramuscular vaccination (IM). In order to identify the best vaccination approach that would overcome the immunotolerant status of pregnancy, we analyzed the potency and duration of protective immunity in vaccinated mothers and their offspring after a single vaccination. Since aluminum salts have been associated with high incidence of local side effects (pain, irritation, inflammation) and may not be suitable for skin vaccination [31–33], we compared efficacy of skin immunization with tetanus toxoid to intramuscular immunization with commercially available Alum® adjuvanted vaccine. 2. Materials and Methods 2.1. Tetanus toxoid and toxin TT monobulk formaldehyde inactivated was kindly provided by the Serum Institute of India (Pune, India). Tetanus toxin from C. tetani (MW 150 kDa) was purchased from Sigma-Aldrich (St. Louis, MO). The preparation has 0.01% thimerosal and 0.9% NaCl. Alum® adjuvanted vaccine (Tetana; IBSS Biomed, Krakow, Poland) was lyophilized and concentrated at 5 Lf per 50 μl for comparison. 2.2. Antigen characterization TT was analyzed by SDS-PAGE on a 10% gel under denaturing conditions and by Western blotting or Coomassie stain (Sigma Aldrich, St Louis, MO) according to standard protocols. The immunoreactive bands were detected with anti-TT HRP-labeled antibodies from the ELISA kit used for TT quantification (#TTX-314, Alpha Diagnostics). Dual color protein standards were obtained from Bio-Rad Laboratories (Hercules, CA). 2.3. Fabrication of MNPs for encapsulation of tetanus toxoid vaccine MNPs were prepared by a two-step micro-molding process described previously [34]. Briefly, TT monobulk was first concentrated ten-fold using 10 kDa MWCO spin filters (EMD Millipore, Billerica, MA). Then, a vaccine casting solution was prepared containing 3% (w/ v) polyvinyl alcohol (PVA; Sigma-Aldrich St Louis, MO),10% (w/v) sucrose (Sigma-Aldrich) and 1% (w/v) CMC (Sigma-Aldrich) in 100 mM dibasic potassium phosphate buffer pH 7.4 to which concentrated TT monobulk was added. In some cases sulforhodamine dye was added to facilitate imaging. This solution was cast onto a PDMS mold (100
conical microneedles per array; each microneedle measuring 650 μm in length and 250 μm in diameter at the base) and exposed to vacuum to facilitate filling the microneedle cavities. When multiple patches were made in a batch, casting solution was applied to all of the molds within 30 s. After 5 min, excess vaccine casting solution was removed from the mold surface and the patches were air-dried overnight at room temperature and for another 12–15 h under vacuum at 37 °C. Careful control to achieve uniform timing of casting solution application and removal from the mold surface facilitated reproducible vaccine loading into patches by minimizing patch-to-patch variation in evaporation of casting solution. Extended drying with heat and vacuum facilitated making stronger microneedle tips, because residual water softens the tips. In the second step, a backing casting solution consisting of 20% PVA and 20% sucrose in 100 mM dibasic potassium phosphate buffer pH 7.4 was cast onto the mold under vacuum and subsequently dried at room temperature overnight before demolding the MNP and storing in aluminum pouches with desiccant at 4 °C before use to vaccinate animals within 24 h after patch fabrication. This two-step fabrication procedure loaded TT vaccine preferentially into the microneedle tips and away from the base substrate. To determine vaccine delivery to skin, MNPs were cut in halves. One half was manually applied to depilated mouse skin in vivo, held in place for 2 min and left on skin for a total of 20 min while the other half was stored until analysis. ELISA (Alpha Diagnostics International, San Antonio, TX) was used to determine the difference in TT content in the used and unused halves, which was taken to equal the amount of TT delivered to the skin. Analysis of swabs wiped on the skin surface after MNP application indicated that very little TT was left on the skin surface (data not shown). 2.4. Animals Eight week-old female and male BALB/c mice (Harlan Laboratories, Dublin, VA) were bred and housed in a biosafety level 1 facility for immunizations. Tetanus challenges took place in BSL2+ level animal facility at Emory University. All experimental protocols involving animal studies that have been carried out under approval of Emory University's IACUC guidelines. 2.5. Breeding protocol Since the BALB/c mouse strain is a poor breeder, we established a harem breeding protocol where four females in proestrus or estrus were housed with one male for 3–4 days [35]. Stages of estrous cycle were determined by visible changes in external genitalia [36]. Female mice were observed daily for the presence of a copulation plug [37] indicative of mating and monitored for body weight changes reflective of pregnancy. 2.6. Determination of lethal tetanus toxin dose 50 (LD50) Age and weight matched mice were divided into groups of five and challenged with tetanus toxin at concentrations of 100, 50, 10, 5, 2, 1, and 0.1 ng protein/50 μl PBS via subcutaneous injection in the dorsal surface of the hind leg. The mice were examined every 12 h for four days for symptoms of paralysis and physical status [38]. Animals exhibiting paralysis of the toxin-injected hind leg which does not function for walking (stage T3 of paralysis) were humanely euthanized according to IACUC guidelines. The experiment was duplicated for calculation of LD50 [39]. 2.7. Immunizations and sample collection Pregnant or non-pregnant females (n = 10–15 per group) received a single dose of TT (5 Lf) intramuscularly (IM), intradermally (ID) or
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with freshly made MNPs. We did not include a negative control group with “blank” MNPs containing no vaccine because previous studies have shown that they induced no antigen-specific immune responses [40,41]. Pregnant mice were vaccinated mid-gestation (11–13 days). For cutaneous immunization, skin was prepared as previously described [42]. MNPs encapsulating TT were inserted into the skin on the caudal region of the dorsum with a force of ~9 lbf and left in place for 20 min to deliver the antigen. For IM vaccinations, TT in 50 μl PBS was injected into the upper quadrant of the hind leg. For ID vaccinations, TT in 30 μl of PBS was injected with a tuberculin syringe on the same site of MNP insertion. The formation of a bleb would prove successful antigen delivery. Serum was collected from adult mice via submandibular bleeding at weeks 1, 2 and 4 post-immunization. The offspring of pregnant mice were bled at weeks 3, 4, 5, 6, 8, 10, and 12 from birth. 2.8. Mouse challenge studies with tetanus toxin At day 28 post-immunization vaccinated and naïve adult mice were bled and 2 days later they were challenged with 50xLD50 of tetanus toxin. Three week-old pups were challenged with a lower lethal dose (8.33xLD50) due to their low body weight and young age. Challenged mice were monitored for disease progression (stages of paresis/paralysis and body weight changes) for 4 days. All animal studies had approval of Emory University's Institutional Animal Care and Use Committee. 2.9. Evaluation of humoral immune responses
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converted HAI and NT titers were analyzed with one or two way ANOVA with Bonferroni post-test. Unless otherwise stated the antibody assays were at least duplicated. For survival curves, statistics were calculated using a Log-rank (Mantel-Cox) test. Non-linear regression analyses were performed to determine the IC50 (95% confidence interval). A p value b 0.05 was considered significant.
3. Results 3.1. Preservation of tetanus toxoid integrity The molecular weight (MW) of tetanus toxin is about 150 kDa, but the detoxified TT contains a mixture of monomer, dimers and oligomers formed during the preparation process [50,51] with molecular sizes ranging from 150 kDa to over 200 kDa [52]. As shown in Fig. 1 the antigen had a MW ranging from 100 to 250 kDa in agreement with previously reported TT vaccine compositions [52]. The unconcentrated toxoid maintained stability when stored at 4 °C or − 20 °C up to 10 months; when stored at 25 °C for 3 days it showed 11% degradation (data not shown). More than 95% of antigen was retained above the 10 kDa MWCO membrane during ultrafiltration (data not shown). The average content of unconcentrated TT encapsulated MNPs was 0.69 ± 0.1 Lf (n = 4). To increase TT loading, MNPs were fabricated with 10 ×-concentrated TT, which had 6.1 ± 1.1 Lf (n = 3) each, the amount being consistent with the required dose for human vaccines (≥5 Lf) delivered intramuscularly [53].
Anti-TT specific antibody levels were determined quantitatively by ELISA [43]. Nunc Maxisorb plates were coated with 100 ng TT (Enzo Life Sciences, Inc., Farmingdale, NY) per well to capture TT-specific antibodies. Total IgG and the IgG1 and IgG2a isotypes were quantified from the standard curves generated with appropriate purified mouse immunoglobulins and isotype-specific HRP-labeled detection antibodies (Southern Biotech, Birmingham, AL). Functional antibody titers were determined with an in vivo tetanus toxin neutralization assay [44,45]. Briefly, for each group, we pooled equal amounts of sera from all animals in a group and prepared serial two-fold dilutions in PBS. Serum dilutions were incubated overnight at + 4 °C with equal volume of tetanus toxin (dosages: 5xLD50 for adults and 2xLD50 for young). Each mixture was subcutaneously injected into a single naïve mouse and symptoms of tetanus-induced paralysis were recorded over a 4-day period. Titers were established as the inverse serum dilutions that demonstrated protection against infection. 2.10. Measurement of the avidity of anti-TT antibodies TT-specific antibody avidity was measured by ELISA in the presence of increasing concentrations (0–2.0 M) of the chaotropic agent guanidine thiocyanate (GTC) (Sigma; St. Louis, MO) [46–49]. Pooled diluted serum from each group (OD ~ 1) was added in duplicates to the plates and incubated at 1.5 h at 37 °C followed by addition of GTC. At the end of the incubation period, the remaining bound antibodies were detected by goat anti-mouse IgG-HRP for 1 h at 37 °C followed by colorimetric reaction with O-phenylenediamine (Life Technologies, Frederick, MD). The plates were read at 490 nm using the xMark™ microplate absorbance spectrophotometer (Bio-Rad, Hercules, CA). The percentage of bound antibody was determined by comparing the absorbance values measured in the presence of GTC treatment (0.25, 0.5, 1.0, 1.5, and 2.0 M) with that in the absence of GTC treatment. The avidity index was determined by calculating the molar concentration of GTC required to decrease the initial optical density by 50% using Prizm Software. 2.11. Statistics For ELISAs, the statistical significance of differences between two groups was calculated by two-tailed unpaired Student's t-test. Log2
Fig. 1. Composition of tetanus toxoid and encapsulation into MNPs. TT monobulk was analyzed by SDS-PAGE followed by (A) Coomassie stain and (B) Western blotting. Lane 1; Protein standards with the masses indicated on the left of lane 1 A. Lane 2: TT, 7.5 g total protein. The immunoreactive band in Western blot was detected with anti-tetanus toxoid-labeled antibodies.
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3.2. MNPs showed successful skin penetration and antigen release MNPs containing TT were prepared with sulforhodamine as well in order to visualize the geometry of microneedles (Fig. 2A) To increase TT delivery into the skin, the TT was concentrated at the microneedle tip (Fig. 2B) using a two-step fabrication process that filled vaccine casting solution into the microneedle mold cavities and then applied backing casting solution (containing no vaccine) to form the patch backing (see Methods). Both low-TT content (unconcentrated TT in casting solution) (Fig. 2B) and high–TT content (10× concentrated TT in casting solution) (Fig. 2C), MNPs showed successful penetration and complete dissolution in mouse skin within 20 min, leaving a clear insertion pattern on skin post-application (Fig. 2D–F). Patches initially loaded with 10 Lf of TT had a residual material of 0.625 ± 0.695 Lf (average ± standard deviation, n = 6) after insertion in the skin, indicating an excellent delivery efficiency.
3.4. Pregnant mice have lower humoral immune responses than non-pregnant mice The levels of serum TT-specific antibodies produced by vaccinated mice revealed that irrespective of immunization route the non-pregnant cohort developed more robust humoral immune responses than the respective pregnant cohort. A single dose of TT delivered intramuscularly in pregnant mice produced only 8% of serum TT-specific IgG responses and 27% of IgG1 responses compared with those seen in nonpregnant mice (Fig. 3A, B). Pregnant mice vaccinated with MNPs, intradermally, or intramuscularly with Alum® fared better, but still had only 49%–65% of the IgG responses and 36% to 60% of the IgG1 responses compared to non-pregnant mice. These results show that pregnancy suppresses the immune response. 3.5. MNP immunization elicits superior antibody responses to intradermal or intramuscular vaccination
3.3. Timing of pregnancies The identification of stages of the estrous cycle and the establishment of a modified harem breeding protocol resulted in an increase of breeding efficiency and accurate determination of the gestational age. Since the estrous cycle is very short (4–5 days) observation of external genitalia was a quick and less labor intensive approach than vaginal cytology to identify each stage. Since copulation plugs were observed in only 50% of pregnant mice, timing of pregnancies was based on body weight increases of approximately 20% between days 11–13 after mating. Following immunization, body weights continued to be recorded until delivery. No adverse effects of IM or MNP vaccination on pregnancy were observed (i.e. body weight fluctuations or preterm labor).
One month post-MNP vaccination, we detected up to 4-fold increase in IgG titers in the non-pregnant population (Fig. 3A) and up to 9-fold increase in IgG1 titers when compared to IM and ID routes (p b 0.001) (Fig. 3B) whereas the differences between MNP and IM or ID vaccination were not-significantly different in IgG2a production (Fig. 3C). MNP immunization was also found to be the most successful approach in the pregnant cohort as demonstrated by the robust IgG antibody production detected as early as day 7 post-prime, reaching 3-fold and 23fold higher IgG levels than the ID and IM groups respectively one month later (p b 0.001) (Fig. 3D). A similar trend was observed in TTspecific IgG1 and IgG2a antibody production which was enhanced 5fold and 11-fold respectively in the MN vaccinated mice when
Fig. 2. Fabrication of MNPs with tetanus toxoid. (A) MNPs were loaded with TT and sulforhodamine B dye to facilitate imaging. TT was loaded on the microneedle mold using (B) 1× and (C) 10× concentrated TT formulation. (D) Microneedles with dye before skin insertion. (E) Microneedles 20 min after insertion into murine skin ex vivo. (F) Murine skin after removal of MNP, showing deposition of dye from the microneedles in the skin.
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Fig. 3. Humoral immune responses in mice immunized with Tetanus Toxoid. Non-pregnant (A–C) and pregnant (D–F), adult BALB/c mice were immunized with 5 Lf of tetanus toxoid via skin and intramuscular vaccination. Serum samples were collected at 7, 14, and 28 days post-immunization. Total serum IgG titers (A & D), and the IgG isotypes, IgG1 (B & E) and IgG2a (C & F) titers were analyzed against tetanus toxoid by quantitative ELISA. Values are expressed as mean ± SEM. Alum®-IM: Alum®-adjuvanted intramuscularly immunized group (n = 10), MN: non-adjuvanted MNP-immunized group, ID: non-adjuvanted intradermally immunized group, IM: non-adjuvanted intramuscularly immunized group, N: naïve (n = 5). MN, IM and ID groups, n = 5 for non-pregnant mice and n = 10 for pregnant mice.
compared to the ID and IM groups (Fig. 3E, F). Overall, MNP delivery of TT reduced the TT-specific IgG differences between pregnant and nonpregnant groups by 6-fold. Importantly, it improved the IgG2a responses in pregnant mice 6-fold when compared to IM vaccination. Taken together these data demonstrate that the state of pregnancy affects the magnitude and quality of humoral immune responses and that MNP delivery enhances these responses. TT MNP vaccination of non-pregnant mice was not found to be inferior to IM vaccination with Alum® TT. However, in the pregnant cohort the MNP group showed 40% lower TT-specific IgG titers than the Alum®-IM group (P b 0.001, Fig. 3). This difference was mainly attributed to the marked decrease of IgG1 (p b 0.001) and not the IgG2a antibodies (p N 0.05). 3.6. Avidity of tetanus antibodies is equivalent in pregnant mice immunized via MNPs and Alum®-adjuvanted vaccine Although IM vaccination with Alum®-TT induced higher levels of IgG antibodies than MNP vaccination with unadjuvanted TT in pregnant mice, the quality of elicited antibodies as determined with avidity
studies was equivalent. The antibody avidity in the non-pregnant cohort was highest in the Alum®-IM group (IC50 0.9643), followed by the MNP group (IC50 0.8523). The ID (IC50 0.6713) and IM (IC50 0.5818) groups were both significantly lower than the Alum®-IM group (p b 0.001) (Fig. 4A, Suppl. Table I). In the pregnant cohort, the MN group demonstrated improved avidity to the Alum®-IM group (IC50 0.0 9456 vs 0.8945) as well the ID and IM groups (Fig. 4B, Suppl. Table I). The results suggest that skin immunization with MNPs induces the same quality if not the magnitude of humoral immune responses in the immunotolerant state of pregnancy. 3.7. MNPs induce increased tetanus-specific neutralizing antibody titers Analysis of tetanus-specific neutralizing antibodies showed that the lowest titers were elicited in the IM group and the highest titers in the MNP group in both pregnant and non-pregnant cohorts. Although there were no differences in the titers of pregnant and non-pregnant mice immunized intradermally or intramuscularly, the MNP vaccinated and the alum-IM non-pregnant mice had 2-fold higher titers than the pregnant mice (Fig. 4C,D). The neutralizing antibodies demonstrated a
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Fig. 4. Functional antibodies and protective immunity. Serum antibody avidity was determined with chaotropic ELISA using guanidine thiocyanate in (A) non-pregnant and (B) pregnant mice 28 days post-vaccination with TT. Neutralizing antibody titers were assessed via an in vivo neutralization assay in pooled sera of (C) non-pregnant and (D) pregnant groups 28 days post-vaccination. Survival curves of (E) non-pregnant (n = 5 per group) and (F) pregnant mice (n = 10 for ID and IM; n = 13 for MN, n = 5 for naïve and Alum-IM mice) were recorded after challenge with 50xLD50 tetanus toxin. Abbreviations as listed in the legend for Fig. 3.
strong correlation with the serum binding antibodies, particularly for the pregnant state (Suppl. Fig. 1A, B), suggesting that either method can be used accurately as a correlate of immunity in the mouse model [54].
3.8. Cutaneous vaccination with MNPs is uniquely able to confer complete protection against high lethal challenge dose of tetanus toxin All MNP vaccinated mice exhibited superior protection against challenge with a 50xLD50 tetanus toxin dose one month after receiving TT. A 100% survival rate was observed in Alum-IM (p = 0.033) and ID vaccinated (p = 0.05) non-pregnant mice significantly higher than the IM group which showed only partial survival (40%) (Fig. 4E). Although the serum TT-specific antibody titers elicited in the pregnant population by MNP or ID vaccination were at least 50% lower than those observed in the non-pregnant population, protection was higher than expected, with survivals rates of 84% and 70% respectively. In contrast, most intramuscularly immunized mice did not survive challenge showing only 20% protection and being significantly lower than MNPs (p = 0.001) or ID immunized groups (p = 0.013) (Fig. 4F). Both Alum-IM non-pregnant and pregnant mice survived 100% although only the latter group
showed statistically significant differences from the unadjuvanted IM control (p = 0.015). The survival rates correlated strongly with the neutralizing antibody titers (NT) in pregnant vaccinated mice (R2, 0.9845) (Suppl. Fig. 1D). A titer of 10 for the ID group was indicative of protection with a survival rate at 70% whereas a titer of 20 increased the survival rate to 84%. The correlation was weaker between neutralizing antibodies and survival in the non-pregnant population (0.4902) since a titer of 5 in the IM group corresponded to 40% protection whereas in the pregnant population the same titer corresponded to only 20% protection (Suppl. Fig. 1C). Mice with antibody titers above 10 were fully protected as shown in both the ID and MN vaccinated groups. These data suggest that strong protection of the non-pregnant population does not depend solely on neutralizing antibodies whereas pregnant mice rely mostly on humoral responses for protection against a lethal challenge dose of tetanus toxin. 3.9. Passive immunity is more robust in the offspring of MNP-vaccinated mothers Sera of offspring born to mothers vaccinated during pregnancy were analyzed at set time points until week 12 post-birth for TT-specific antibodies. At the end of weaning period (week 3), the antigen-specific IgG
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titers were 5 to 10-fold higher in the MNP group when compared to ID and IM groups respectively (p b 0.001); They were detectable until week 12, longer than the ID and IM groups indicating a greater passive immune response as a result of MNP maternal vaccination (Fig. 5A). Similar differences were observed in IgG1 isotype levels (Fig. 5B) whereas IgG2a antibodies were detected only in offspring of MNP-vaccinated mice up to week 4 (Fig. 5C). As a result, the IgG1/IgG2a ratio varied from 200 in the ID group to 23 in the MNP group suggesting activation of both arms of immunity (Fig. 5D). 3.10. Offspring of MNP-vaccinated mothers are fully protected against lethal challenge with tetanus toxin The protective efficacy of maternal anti-TT IgG antibodies from the placenta or from maternal milk was tested in 3 week-old pups following challenge with 8.3xLD50 of tetanus toxin. The survival rates for the pups born to Alum-IM, MNP, ID and IM mothers were 100%, 100%, 87.5% and 13%, respectively (Fig. 6A) and correlated well with the neutralizing antibodies (0.74) (Suppl. Fig. 1E). A titer of 5 found in offspring from ID immunized mothers was sufficient to confer 87.5% protection against the toxin (Fig. 6B). The correlation between binding antibodies and neutralizing titers was lower in the neonates than in the mothers (R2 = 0.94 vs. 0.99 respectively). Six week old pups from Alum-IM and MNP-immunized mothers were fully protected against lethal infection whereas the offspring from ID and IM immunized mothers showed 20% and 0% survival rates respectively (Fig. 6C). Offspring challenged with the same dose of tetanus toxin 12 weeks after birth did not survive the infection although the pups from MNP immunized mothers delayed developing symptoms of disease until day 4, suggesting a limited degree of protection (Fig. 6D). Statistical analysis of survival rates showed significant differences between MNP and IM vaccination up to week 6 (pweek3 b 0.0001, pweek6 = 0.0003). While there was not a significant difference between MN and ID vaccination at week 3, the former outperformed intradermal vaccination at week 6 (p = 0.008) and week 12 (p = 0.03).
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The data from this experiment confirm the superiority of MNP vaccination, since the passive immunity observed in the offspring of vaccinated mothers was robust and long-lived, with antibodies fully protective at least up to 6 weeks after birth. 4. Discussion In this study, we examined the host immune responses to TT induced by skin delivery and compared them to conventional intramuscular immunization with TT and to commercially available Alum® adjuvanted vaccine administered IM. We developed MNPs encapsulating TT without adjuvant for cutaneous vaccination of pregnant and non-pregnant BALB/c mice and found that a single dose of 5 Lf (flocculation units) delivered with MN was sufficient to induce robust humoral immune responses sufficient to successfully protect the vaccinated animals against a lethal challenge dose with tetanus toxin (50xLD50). Matsuo et al. reported that cutaneous immunization of non-pregnant rats with dissolving MNPs containing unadjuvanted TT induced effective immune responses as well as IM and ID routes of TT delivery after 5 vaccine doses [55]. Our delivery system demonstrated superiority to IM and ID routes in both non-pregnant and pregnant cohorts and their offspring. Immunization with MNPs improved affinity maturation as seen by isotype class switching when compared to ID or IM routes. It also conferred a greater passive immune response amongst offspring as well as significant protection against high challenge dose with tetanus toxin compared to the offspring of mice vaccinated intramuscularly during pregnancy. Insertion of MNPs in the skin causes micro-injury of keratinocytes, releasing cytokines involved in innate cell recruitment in the area of needle insertion and antigen delivery [56]. Thus skin vaccination can exert an adjuvant effect per se potentially replacing the need for an adjuvant necessary to increase the immunogenicity of toxoid. Our findings agree with this hypothesis but only for the non-pregnant population in which MNP administration of TT without adjuvant induced levels of total immunoglobulins similar to IM vaccination of TT with Alum as
Fig. 5. Passive immunity in offspring of vaccinated with tetanus toxoid mice. Passively transferred TT-specific antibodies were determined in serum of offspring from vaccinated mice with ELISA. (A) Total IgG (B) IgG1 (C) IgG2a. Values are expressed as mean ± SEM (n = 5–12 mice per group. (D) IgG1 vs IgG2a antibody ratio was calculated for weeks 3 and 4. Alum IM: offspring of mothers immunized IM with Alum-TT vaccine (n = 5); MN: offspring of mothers immunized with MNPs (n = 10); ID: offspring of mothers immunized intradermally (n = 10); IM: offspring of mothers immunized intramuscularly (n = 10); N: naïve (n = 5).
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Fig. 6. Protective immune responses in offspring of vaccinated mothers. Independent cohorts of offspring born from mothers immunized with 5Lf Tetanus Toxoid using MNPs (MN) (n = 10), intradermal (ID) (n = 10), or intramuscular (IM) injection (n = 10) or immunized IM with Alum-TT vaccine (n = 5) as well as naïve (N) controls (n = 5) were challenged with 8.33xLD50 tetanus toxin at (A) 3-weeks, (C) 6-weeks, and (D) 12-weeks of age and followed up to 4 days for survival. (D) Neutralizing TT-specific antibody titers were assessed in pooled-serum of 3-week old pups. The titer was the highest dilution of pooled serum in PBS that neutralized 2xLD50 Tetanus Toxin. Abbreviations are as listed in the legend for Fig. 5.
adjuvant. Interestingly, although the IM administered Alum® TT vaccine induced almost 2-fold higher IgG responses than the MNPs administered unadjuvanted TT in pregnant mice, the immunoglobulin avidity and affinity were similar between these vaccination routes in both nonpregnant and non-pregnant cohorts and both groups survived challenge with tetanus toxin. The underlying mechanisms need to be further investigated. The potency of passively transferred antibodies in the newborns reflected to a certain extent the differences seen in antibody titers in the mothers. The most significant correlation was identified between neutralizing antibody titers and percent survival in 3 week old newborns. Interestingly, mice from MNP-vaccinated mothers had significantly higher IgG2a titers than neonates from Alum® TT vaccinated mothers whereas these titers were not different in the mothers. These data suggest that this enhanced response may be partly attributed to passively transferred maternal antibodies and partly to the developing immune system of the offspring. They also agree with a recent report from our group showing that young mice vaccinated with influenzacoated metal MNPs had improved IgG2a responses compared to IM vaccinated animals [57]. Two types of effector CD4+ T helper cell responses can be induced by professional antigen presenting cells, designated Th1 and Th2, each designed to eliminate different types of pathogens. Th2 responses are critical for humoral immunity necessary for control of bacterial pathogens (i.e. tetanus) due to neutralizing antibody production by IL-4-activated B cells. While the responses in immunized non-pregnant cohorts were primarily of the Th2 type, as expected by the nature of TT [58], the Th2 bias was significantly less in the pregnant cohort as determined by increased IgG2a production reflective of Th1 type, mainly after MNP vaccination. No changes were observed in the IgG1/IgG2a ratios seen
in Alum-IM vaccinated pregnant and non-pregnant cohorts that demonstrated a clear Th2 bias. It is worth noting that despite the 3-fold difference of TT neutralizing antibody titers between MNP (GMT 80) and ID immunized (GMT 10) non-pregnant mice, both groups fully survived a high lethal challenge whereas neither the MNP (GMT 20) nor the ID (GMT 10) immunized pregnant mice showed full protection against the same dose. These data suggest that antibody quality, which plays a very important role in protective immunity, may be affected at transcriptional or translational levels by pregnancy hormones. Although further product development and human clinical studies are needed, the results of this study demonstrate the potential of MNP vaccination to protect pregnant women and their newborn babies against tetanus. Our aim is to achieve a protective immune response with reduced vaccine doses and elimination of possible reactogenicity of aluminum salts by harnessing the immunological power of skin immunization. 5. Conclusions The first aim of the study was to assess the immune responses to TT elicited in pregnant and non-pregnant mice after intramuscular or cutaneous vaccination with either MNPs or intradermal injection. It was shown that unadjuvanted TT could be encapsulated into MNPs and delivered to murine skin. MNP-vaccination with unadjuvanted TT induced a significantly more robust immune response as well as superior protection to tetanus toxin challenge compared to intramuscular vaccination. The antibody responses were comparable to commercially available Alum® adjuvanted vaccine administered intramuscularly. It was also observed that pregnant mice regardless of their immunization route
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had lower humoral immune responses and were less protected against lethal tetanus toxin challenge than their non-pregnant controls. The second aim of the study was to compare the passive immune responses in the offspring of immunized pregnant mice. The passive immunity was more robust and longer lasting in the offspring of MNPvaccinated mothers. The offspring of Alum® adjuvanted vaccine and MNP vaccinated mothers were conferred complete protection against lethal tetanus toxin challenge at six weeks of age while the offspring of intramuscularly vaccinated mothers without Alum® had no protection. These results in the mouse model have demonstrated the potential of MNP technology to reduce the incidences of maternal and neonatal tetanus using a simple, patch-based delivery system.
[11]
[12]
[13]
[14] [15]
[16]
Research funding The work was supported by United States Agency for International Development (AID-OAA-F-13-00083 award). Conflict of interest Mark Prausnitz is an inventor of patents that have been licensed to companies developing microneedle-based products, is a paid advisor to companies developing microneedle-based products, and is a founder/shareholder of companies developing microneedle-based products (Micron Biomedical). The terms of this arrangement have been reviewed and approved by Georgia Tech and Emory University in accordance with their conflict of interest policies. E. Stein Esser, Richard Compans and Ioanna Skountzou have filed invention disclosure for tetanus skin immunization. Elena V. Vassilieva and Andrey A. Romanyuk, declare that they have no conflict of interest.
[17] [18] [19] [20]
[21]
[22]
[23] [24]
[25]
[26]
Acknowledgements We thank Dahnide Taylor-Williams, Erin-Joi McNeal, Nadia Lelutiu and Donna Bondy for their valuable technical and administrative support. We thank Haripriya Kaluri for her assistance to obtain unadjuvanted tetanus toxoid. We thank Serum Institute of India for providing tetanus toxoid monobulk.
[27]
[28]
[29]
Appendix A. Supplementary data [30]
Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.jconrel.2016.06.026. [31]
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Contents lists available at ScienceDirect
Journal of Controlled Release journal homepage: www.elsevier.com/locate/jconrel
Tetanus vaccination with a dissolving microneedle patch confers protective immune responses in pregnancy E. Stein Esser a, AndreyA. Romanyuk b, Elena V. Vassilieva a, Joshy Jacob a, Mark R. Prausnitz b, Richard W. Compans a, Ioanna Skountzou a,⁎ a b
Department of Microbiology and Immunology and Emory Vaccine Center, Emory University School of Medicine, Atlanta 30322, Georgia School of Chemical and Biomolecular Engineering, Georgia Institute of Technology, Atlanta 30332, Georgia
a r t i c l e
i n f o
Article history: Received 3 December 2015 Received in revised form 24 April 2016 Accepted 16 June 2016 Available online 18 June 2016 Keywords: Dissolving microneedle patches Skin immunization Tetanus toxoid Mouse model Pregnancy Survival
a b s t r a c t Maternal and neonatal tetanus claim tens of thousands lives every year in developing countries, but could be prevented by hygienic practices and improved immunization of pregnant women. This study tested the hypothesis that skin vaccination can overcome the immunologically transformed state of pregnancy and enhance protective immunity to tetanus in mothers and their newborns. To achieve this goal, we developed microneedle patches (MNPs) that efficiently delivered unadjuvanted tetanus toxoid to skin of pregnant mice and demonstrated that this route induced superior immune responses in female mice conferring 100% survival to tetanus toxin challenge when compared to intramuscular vaccination. Mice born to MNP-vaccinated mothers showed detectable tetanus–specific IgG antibodies up to 12 weeks of age and complete protection to tetanus toxin challenge up at 6 weeks of age. In contrast, none of the 6-week old mice born to intramuscularly vaccinated mothers survived challenge. Although pregnant mice vaccinated with unadjuvanted tetanus toxoid had 30% lower IgG and IgG1 titers than mice vaccinated intramuscularly with Alum®-adjuvanted tetanus toxoid vaccine, IgG2a titers and antibody affinity maturation were similar between these groups. We conclude that skin immunization with MNPs containing unadjuvanted tetanus toxoid can confer potent protective efficacy to mothers and their offspring using a delivery method well suited for expanding vaccination coverage in developing countries. © 2016 Elsevier B.V. All rights reserved.
1. Introduction Tetanus is an acute disease caused by a neurotoxin produced by the anaerobic bacterium Clostridium tetani with motor and autonomous nervous system manifestations such as muscle rigidity and convulsive spasms [1]. Despite a 93% reduction of disease prevalence since launching of the Maternal and Neonatal Tetanus Elimination Initiative in 1988, tetanus has yet to be eliminated in 22 countries. In the areas where access to antenatal care is well below 50%, the incidence of neonatal tetanus is around 10% presenting a major public health concern [2–6]. In 2013 alone, material and neonatal tetanus claimed lives of several thousands of women and 50,000 newborns [7]. Once the disease is contracted, the fatality rate can be as high as 100% without hospital care and varies from 10% to 60% even with hospital care [8]. Disease incidence can be reduced through hygienic delivery practices and vaccination with Tdap (adult tetanus, diphtheria, pertussis vaccines). Vaccination with Tdap not only confers protective immunity to the mother, but also provides a passive immunity to the infants prior ⁎ Corresponding author. E-mail address: [email protected] (I. Skountzou).
http://dx.doi.org/10.1016/j.jconrel.2016.06.026 0168-3659/© 2016 Elsevier B.V. All rights reserved.
to the development of their own immune system [9,10]. Irrespective of the patient's prior immunization history, the Tdap vaccine should be administered at any time during pregnancy or immediately postpartum. However, in order to maximize passive antibody transfer, it is recommended that the Tdap vaccine be administered during the last trimester of pregnancy [11]. The current route for tetanus immunization is intramuscular delivery with syringes and needles. This approach presents several immunological and logistical disadvantages. With regards to immunological disadvantages, tetanus toxoid (TT) is a weak antigen [12], hence the addition of an adjuvant (Alum®) as well as repeated boosters are necessary to induce robust antibody titers and affinity maturation [13,14]. Another important problem for effective immunization is the immunotolerant state which results from pregnancy. Unless women have been immunized prior to pregnancy, the recommended immunization does not serve as a booster and only elicits a modest primary response. As for logistical problems to be addressed, particularly in rural areas of developing countries, those include requirement for cold chain, a critical bottleneck for vaccine storage and transportation; distribution and administration by trained personnel. Unfortunately the costs for their implementation are a limiting factor for effective
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administration and vaccination coverage. Thus, in rural areas it is difficult to complete the vaccination regimen; hence one or two tetanus vaccine doses as recommended by WHO are not sufficient to elicit effective responses in a population without pre-existing immunity [15,16]. Therefore new vaccination and administration approaches are urgently needed. Skin is recently gaining more attention as a vaccine target organ due to its immunological properties, which include an increased number of professional antigen presenting cells (APCs) compared to the muscle [17–19]. Antigen uptake initiates a cascade of innate immune responses resulting in its efficient presentation to naïve T and B cells that reside in draining lymph nodes proximal to the insertion site. Effective cross-talk between innate and adaptive immune responses generates more robust, longer-lasting immune protection [20,21]. We and other investigators have previously reported that skin immunization with metal or dissolving microneedle patches (MNPs) carrying various vaccines (influenza, inactivated poliovirus (IPV), measles, tetanus, HPV, rotavirus, BCG, malaria and RSV) confers immune responses at least non-inferior to conventional immunization as shown by lethal challenge studies, longevity of immune responses and breadth of immunity in a number of small-animal models [21–30]. In this study, we determined the immunological advantages of skin tetanus vaccination in pregnancy using MNPs or intradermal injection (ID) in mice and compared the results to intramuscular vaccination (IM). In order to identify the best vaccination approach that would overcome the immunotolerant status of pregnancy, we analyzed the potency and duration of protective immunity in vaccinated mothers and their offspring after a single vaccination. Since aluminum salts have been associated with high incidence of local side effects (pain, irritation, inflammation) and may not be suitable for skin vaccination [31–33], we compared efficacy of skin immunization with tetanus toxoid to intramuscular immunization with commercially available Alum® adjuvanted vaccine. 2. Materials and Methods 2.1. Tetanus toxoid and toxin TT monobulk formaldehyde inactivated was kindly provided by the Serum Institute of India (Pune, India). Tetanus toxin from C. tetani (MW 150 kDa) was purchased from Sigma-Aldrich (St. Louis, MO). The preparation has 0.01% thimerosal and 0.9% NaCl. Alum® adjuvanted vaccine (Tetana; IBSS Biomed, Krakow, Poland) was lyophilized and concentrated at 5 Lf per 50 μl for comparison. 2.2. Antigen characterization TT was analyzed by SDS-PAGE on a 10% gel under denaturing conditions and by Western blotting or Coomassie stain (Sigma Aldrich, St Louis, MO) according to standard protocols. The immunoreactive bands were detected with anti-TT HRP-labeled antibodies from the ELISA kit used for TT quantification (#TTX-314, Alpha Diagnostics). Dual color protein standards were obtained from Bio-Rad Laboratories (Hercules, CA). 2.3. Fabrication of MNPs for encapsulation of tetanus toxoid vaccine MNPs were prepared by a two-step micro-molding process described previously [34]. Briefly, TT monobulk was first concentrated ten-fold using 10 kDa MWCO spin filters (EMD Millipore, Billerica, MA). Then, a vaccine casting solution was prepared containing 3% (w/ v) polyvinyl alcohol (PVA; Sigma-Aldrich St Louis, MO),10% (w/v) sucrose (Sigma-Aldrich) and 1% (w/v) CMC (Sigma-Aldrich) in 100 mM dibasic potassium phosphate buffer pH 7.4 to which concentrated TT monobulk was added. In some cases sulforhodamine dye was added to facilitate imaging. This solution was cast onto a PDMS mold (100
conical microneedles per array; each microneedle measuring 650 μm in length and 250 μm in diameter at the base) and exposed to vacuum to facilitate filling the microneedle cavities. When multiple patches were made in a batch, casting solution was applied to all of the molds within 30 s. After 5 min, excess vaccine casting solution was removed from the mold surface and the patches were air-dried overnight at room temperature and for another 12–15 h under vacuum at 37 °C. Careful control to achieve uniform timing of casting solution application and removal from the mold surface facilitated reproducible vaccine loading into patches by minimizing patch-to-patch variation in evaporation of casting solution. Extended drying with heat and vacuum facilitated making stronger microneedle tips, because residual water softens the tips. In the second step, a backing casting solution consisting of 20% PVA and 20% sucrose in 100 mM dibasic potassium phosphate buffer pH 7.4 was cast onto the mold under vacuum and subsequently dried at room temperature overnight before demolding the MNP and storing in aluminum pouches with desiccant at 4 °C before use to vaccinate animals within 24 h after patch fabrication. This two-step fabrication procedure loaded TT vaccine preferentially into the microneedle tips and away from the base substrate. To determine vaccine delivery to skin, MNPs were cut in halves. One half was manually applied to depilated mouse skin in vivo, held in place for 2 min and left on skin for a total of 20 min while the other half was stored until analysis. ELISA (Alpha Diagnostics International, San Antonio, TX) was used to determine the difference in TT content in the used and unused halves, which was taken to equal the amount of TT delivered to the skin. Analysis of swabs wiped on the skin surface after MNP application indicated that very little TT was left on the skin surface (data not shown). 2.4. Animals Eight week-old female and male BALB/c mice (Harlan Laboratories, Dublin, VA) were bred and housed in a biosafety level 1 facility for immunizations. Tetanus challenges took place in BSL2+ level animal facility at Emory University. All experimental protocols involving animal studies that have been carried out under approval of Emory University's IACUC guidelines. 2.5. Breeding protocol Since the BALB/c mouse strain is a poor breeder, we established a harem breeding protocol where four females in proestrus or estrus were housed with one male for 3–4 days [35]. Stages of estrous cycle were determined by visible changes in external genitalia [36]. Female mice were observed daily for the presence of a copulation plug [37] indicative of mating and monitored for body weight changes reflective of pregnancy. 2.6. Determination of lethal tetanus toxin dose 50 (LD50) Age and weight matched mice were divided into groups of five and challenged with tetanus toxin at concentrations of 100, 50, 10, 5, 2, 1, and 0.1 ng protein/50 μl PBS via subcutaneous injection in the dorsal surface of the hind leg. The mice were examined every 12 h for four days for symptoms of paralysis and physical status [38]. Animals exhibiting paralysis of the toxin-injected hind leg which does not function for walking (stage T3 of paralysis) were humanely euthanized according to IACUC guidelines. The experiment was duplicated for calculation of LD50 [39]. 2.7. Immunizations and sample collection Pregnant or non-pregnant females (n = 10–15 per group) received a single dose of TT (5 Lf) intramuscularly (IM), intradermally (ID) or
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with freshly made MNPs. We did not include a negative control group with “blank” MNPs containing no vaccine because previous studies have shown that they induced no antigen-specific immune responses [40,41]. Pregnant mice were vaccinated mid-gestation (11–13 days). For cutaneous immunization, skin was prepared as previously described [42]. MNPs encapsulating TT were inserted into the skin on the caudal region of the dorsum with a force of ~9 lbf and left in place for 20 min to deliver the antigen. For IM vaccinations, TT in 50 μl PBS was injected into the upper quadrant of the hind leg. For ID vaccinations, TT in 30 μl of PBS was injected with a tuberculin syringe on the same site of MNP insertion. The formation of a bleb would prove successful antigen delivery. Serum was collected from adult mice via submandibular bleeding at weeks 1, 2 and 4 post-immunization. The offspring of pregnant mice were bled at weeks 3, 4, 5, 6, 8, 10, and 12 from birth. 2.8. Mouse challenge studies with tetanus toxin At day 28 post-immunization vaccinated and naïve adult mice were bled and 2 days later they were challenged with 50xLD50 of tetanus toxin. Three week-old pups were challenged with a lower lethal dose (8.33xLD50) due to their low body weight and young age. Challenged mice were monitored for disease progression (stages of paresis/paralysis and body weight changes) for 4 days. All animal studies had approval of Emory University's Institutional Animal Care and Use Committee. 2.9. Evaluation of humoral immune responses
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converted HAI and NT titers were analyzed with one or two way ANOVA with Bonferroni post-test. Unless otherwise stated the antibody assays were at least duplicated. For survival curves, statistics were calculated using a Log-rank (Mantel-Cox) test. Non-linear regression analyses were performed to determine the IC50 (95% confidence interval). A p value b 0.05 was considered significant.
3. Results 3.1. Preservation of tetanus toxoid integrity The molecular weight (MW) of tetanus toxin is about 150 kDa, but the detoxified TT contains a mixture of monomer, dimers and oligomers formed during the preparation process [50,51] with molecular sizes ranging from 150 kDa to over 200 kDa [52]. As shown in Fig. 1 the antigen had a MW ranging from 100 to 250 kDa in agreement with previously reported TT vaccine compositions [52]. The unconcentrated toxoid maintained stability when stored at 4 °C or − 20 °C up to 10 months; when stored at 25 °C for 3 days it showed 11% degradation (data not shown). More than 95% of antigen was retained above the 10 kDa MWCO membrane during ultrafiltration (data not shown). The average content of unconcentrated TT encapsulated MNPs was 0.69 ± 0.1 Lf (n = 4). To increase TT loading, MNPs were fabricated with 10 ×-concentrated TT, which had 6.1 ± 1.1 Lf (n = 3) each, the amount being consistent with the required dose for human vaccines (≥5 Lf) delivered intramuscularly [53].
Anti-TT specific antibody levels were determined quantitatively by ELISA [43]. Nunc Maxisorb plates were coated with 100 ng TT (Enzo Life Sciences, Inc., Farmingdale, NY) per well to capture TT-specific antibodies. Total IgG and the IgG1 and IgG2a isotypes were quantified from the standard curves generated with appropriate purified mouse immunoglobulins and isotype-specific HRP-labeled detection antibodies (Southern Biotech, Birmingham, AL). Functional antibody titers were determined with an in vivo tetanus toxin neutralization assay [44,45]. Briefly, for each group, we pooled equal amounts of sera from all animals in a group and prepared serial two-fold dilutions in PBS. Serum dilutions were incubated overnight at + 4 °C with equal volume of tetanus toxin (dosages: 5xLD50 for adults and 2xLD50 for young). Each mixture was subcutaneously injected into a single naïve mouse and symptoms of tetanus-induced paralysis were recorded over a 4-day period. Titers were established as the inverse serum dilutions that demonstrated protection against infection. 2.10. Measurement of the avidity of anti-TT antibodies TT-specific antibody avidity was measured by ELISA in the presence of increasing concentrations (0–2.0 M) of the chaotropic agent guanidine thiocyanate (GTC) (Sigma; St. Louis, MO) [46–49]. Pooled diluted serum from each group (OD ~ 1) was added in duplicates to the plates and incubated at 1.5 h at 37 °C followed by addition of GTC. At the end of the incubation period, the remaining bound antibodies were detected by goat anti-mouse IgG-HRP for 1 h at 37 °C followed by colorimetric reaction with O-phenylenediamine (Life Technologies, Frederick, MD). The plates were read at 490 nm using the xMark™ microplate absorbance spectrophotometer (Bio-Rad, Hercules, CA). The percentage of bound antibody was determined by comparing the absorbance values measured in the presence of GTC treatment (0.25, 0.5, 1.0, 1.5, and 2.0 M) with that in the absence of GTC treatment. The avidity index was determined by calculating the molar concentration of GTC required to decrease the initial optical density by 50% using Prizm Software. 2.11. Statistics For ELISAs, the statistical significance of differences between two groups was calculated by two-tailed unpaired Student's t-test. Log2
Fig. 1. Composition of tetanus toxoid and encapsulation into MNPs. TT monobulk was analyzed by SDS-PAGE followed by (A) Coomassie stain and (B) Western blotting. Lane 1; Protein standards with the masses indicated on the left of lane 1 A. Lane 2: TT, 7.5 g total protein. The immunoreactive band in Western blot was detected with anti-tetanus toxoid-labeled antibodies.
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3.2. MNPs showed successful skin penetration and antigen release MNPs containing TT were prepared with sulforhodamine as well in order to visualize the geometry of microneedles (Fig. 2A) To increase TT delivery into the skin, the TT was concentrated at the microneedle tip (Fig. 2B) using a two-step fabrication process that filled vaccine casting solution into the microneedle mold cavities and then applied backing casting solution (containing no vaccine) to form the patch backing (see Methods). Both low-TT content (unconcentrated TT in casting solution) (Fig. 2B) and high–TT content (10× concentrated TT in casting solution) (Fig. 2C), MNPs showed successful penetration and complete dissolution in mouse skin within 20 min, leaving a clear insertion pattern on skin post-application (Fig. 2D–F). Patches initially loaded with 10 Lf of TT had a residual material of 0.625 ± 0.695 Lf (average ± standard deviation, n = 6) after insertion in the skin, indicating an excellent delivery efficiency.
3.4. Pregnant mice have lower humoral immune responses than non-pregnant mice The levels of serum TT-specific antibodies produced by vaccinated mice revealed that irrespective of immunization route the non-pregnant cohort developed more robust humoral immune responses than the respective pregnant cohort. A single dose of TT delivered intramuscularly in pregnant mice produced only 8% of serum TT-specific IgG responses and 27% of IgG1 responses compared with those seen in nonpregnant mice (Fig. 3A, B). Pregnant mice vaccinated with MNPs, intradermally, or intramuscularly with Alum® fared better, but still had only 49%–65% of the IgG responses and 36% to 60% of the IgG1 responses compared to non-pregnant mice. These results show that pregnancy suppresses the immune response. 3.5. MNP immunization elicits superior antibody responses to intradermal or intramuscular vaccination
3.3. Timing of pregnancies The identification of stages of the estrous cycle and the establishment of a modified harem breeding protocol resulted in an increase of breeding efficiency and accurate determination of the gestational age. Since the estrous cycle is very short (4–5 days) observation of external genitalia was a quick and less labor intensive approach than vaginal cytology to identify each stage. Since copulation plugs were observed in only 50% of pregnant mice, timing of pregnancies was based on body weight increases of approximately 20% between days 11–13 after mating. Following immunization, body weights continued to be recorded until delivery. No adverse effects of IM or MNP vaccination on pregnancy were observed (i.e. body weight fluctuations or preterm labor).
One month post-MNP vaccination, we detected up to 4-fold increase in IgG titers in the non-pregnant population (Fig. 3A) and up to 9-fold increase in IgG1 titers when compared to IM and ID routes (p b 0.001) (Fig. 3B) whereas the differences between MNP and IM or ID vaccination were not-significantly different in IgG2a production (Fig. 3C). MNP immunization was also found to be the most successful approach in the pregnant cohort as demonstrated by the robust IgG antibody production detected as early as day 7 post-prime, reaching 3-fold and 23fold higher IgG levels than the ID and IM groups respectively one month later (p b 0.001) (Fig. 3D). A similar trend was observed in TTspecific IgG1 and IgG2a antibody production which was enhanced 5fold and 11-fold respectively in the MN vaccinated mice when
Fig. 2. Fabrication of MNPs with tetanus toxoid. (A) MNPs were loaded with TT and sulforhodamine B dye to facilitate imaging. TT was loaded on the microneedle mold using (B) 1× and (C) 10× concentrated TT formulation. (D) Microneedles with dye before skin insertion. (E) Microneedles 20 min after insertion into murine skin ex vivo. (F) Murine skin after removal of MNP, showing deposition of dye from the microneedles in the skin.
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Fig. 3. Humoral immune responses in mice immunized with Tetanus Toxoid. Non-pregnant (A–C) and pregnant (D–F), adult BALB/c mice were immunized with 5 Lf of tetanus toxoid via skin and intramuscular vaccination. Serum samples were collected at 7, 14, and 28 days post-immunization. Total serum IgG titers (A & D), and the IgG isotypes, IgG1 (B & E) and IgG2a (C & F) titers were analyzed against tetanus toxoid by quantitative ELISA. Values are expressed as mean ± SEM. Alum®-IM: Alum®-adjuvanted intramuscularly immunized group (n = 10), MN: non-adjuvanted MNP-immunized group, ID: non-adjuvanted intradermally immunized group, IM: non-adjuvanted intramuscularly immunized group, N: naïve (n = 5). MN, IM and ID groups, n = 5 for non-pregnant mice and n = 10 for pregnant mice.
compared to the ID and IM groups (Fig. 3E, F). Overall, MNP delivery of TT reduced the TT-specific IgG differences between pregnant and nonpregnant groups by 6-fold. Importantly, it improved the IgG2a responses in pregnant mice 6-fold when compared to IM vaccination. Taken together these data demonstrate that the state of pregnancy affects the magnitude and quality of humoral immune responses and that MNP delivery enhances these responses. TT MNP vaccination of non-pregnant mice was not found to be inferior to IM vaccination with Alum® TT. However, in the pregnant cohort the MNP group showed 40% lower TT-specific IgG titers than the Alum®-IM group (P b 0.001, Fig. 3). This difference was mainly attributed to the marked decrease of IgG1 (p b 0.001) and not the IgG2a antibodies (p N 0.05). 3.6. Avidity of tetanus antibodies is equivalent in pregnant mice immunized via MNPs and Alum®-adjuvanted vaccine Although IM vaccination with Alum®-TT induced higher levels of IgG antibodies than MNP vaccination with unadjuvanted TT in pregnant mice, the quality of elicited antibodies as determined with avidity
studies was equivalent. The antibody avidity in the non-pregnant cohort was highest in the Alum®-IM group (IC50 0.9643), followed by the MNP group (IC50 0.8523). The ID (IC50 0.6713) and IM (IC50 0.5818) groups were both significantly lower than the Alum®-IM group (p b 0.001) (Fig. 4A, Suppl. Table I). In the pregnant cohort, the MN group demonstrated improved avidity to the Alum®-IM group (IC50 0.0 9456 vs 0.8945) as well the ID and IM groups (Fig. 4B, Suppl. Table I). The results suggest that skin immunization with MNPs induces the same quality if not the magnitude of humoral immune responses in the immunotolerant state of pregnancy. 3.7. MNPs induce increased tetanus-specific neutralizing antibody titers Analysis of tetanus-specific neutralizing antibodies showed that the lowest titers were elicited in the IM group and the highest titers in the MNP group in both pregnant and non-pregnant cohorts. Although there were no differences in the titers of pregnant and non-pregnant mice immunized intradermally or intramuscularly, the MNP vaccinated and the alum-IM non-pregnant mice had 2-fold higher titers than the pregnant mice (Fig. 4C,D). The neutralizing antibodies demonstrated a
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Fig. 4. Functional antibodies and protective immunity. Serum antibody avidity was determined with chaotropic ELISA using guanidine thiocyanate in (A) non-pregnant and (B) pregnant mice 28 days post-vaccination with TT. Neutralizing antibody titers were assessed via an in vivo neutralization assay in pooled sera of (C) non-pregnant and (D) pregnant groups 28 days post-vaccination. Survival curves of (E) non-pregnant (n = 5 per group) and (F) pregnant mice (n = 10 for ID and IM; n = 13 for MN, n = 5 for naïve and Alum-IM mice) were recorded after challenge with 50xLD50 tetanus toxin. Abbreviations as listed in the legend for Fig. 3.
strong correlation with the serum binding antibodies, particularly for the pregnant state (Suppl. Fig. 1A, B), suggesting that either method can be used accurately as a correlate of immunity in the mouse model [54].
3.8. Cutaneous vaccination with MNPs is uniquely able to confer complete protection against high lethal challenge dose of tetanus toxin All MNP vaccinated mice exhibited superior protection against challenge with a 50xLD50 tetanus toxin dose one month after receiving TT. A 100% survival rate was observed in Alum-IM (p = 0.033) and ID vaccinated (p = 0.05) non-pregnant mice significantly higher than the IM group which showed only partial survival (40%) (Fig. 4E). Although the serum TT-specific antibody titers elicited in the pregnant population by MNP or ID vaccination were at least 50% lower than those observed in the non-pregnant population, protection was higher than expected, with survivals rates of 84% and 70% respectively. In contrast, most intramuscularly immunized mice did not survive challenge showing only 20% protection and being significantly lower than MNPs (p = 0.001) or ID immunized groups (p = 0.013) (Fig. 4F). Both Alum-IM non-pregnant and pregnant mice survived 100% although only the latter group
showed statistically significant differences from the unadjuvanted IM control (p = 0.015). The survival rates correlated strongly with the neutralizing antibody titers (NT) in pregnant vaccinated mice (R2, 0.9845) (Suppl. Fig. 1D). A titer of 10 for the ID group was indicative of protection with a survival rate at 70% whereas a titer of 20 increased the survival rate to 84%. The correlation was weaker between neutralizing antibodies and survival in the non-pregnant population (0.4902) since a titer of 5 in the IM group corresponded to 40% protection whereas in the pregnant population the same titer corresponded to only 20% protection (Suppl. Fig. 1C). Mice with antibody titers above 10 were fully protected as shown in both the ID and MN vaccinated groups. These data suggest that strong protection of the non-pregnant population does not depend solely on neutralizing antibodies whereas pregnant mice rely mostly on humoral responses for protection against a lethal challenge dose of tetanus toxin. 3.9. Passive immunity is more robust in the offspring of MNP-vaccinated mothers Sera of offspring born to mothers vaccinated during pregnancy were analyzed at set time points until week 12 post-birth for TT-specific antibodies. At the end of weaning period (week 3), the antigen-specific IgG
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titers were 5 to 10-fold higher in the MNP group when compared to ID and IM groups respectively (p b 0.001); They were detectable until week 12, longer than the ID and IM groups indicating a greater passive immune response as a result of MNP maternal vaccination (Fig. 5A). Similar differences were observed in IgG1 isotype levels (Fig. 5B) whereas IgG2a antibodies were detected only in offspring of MNP-vaccinated mice up to week 4 (Fig. 5C). As a result, the IgG1/IgG2a ratio varied from 200 in the ID group to 23 in the MNP group suggesting activation of both arms of immunity (Fig. 5D). 3.10. Offspring of MNP-vaccinated mothers are fully protected against lethal challenge with tetanus toxin The protective efficacy of maternal anti-TT IgG antibodies from the placenta or from maternal milk was tested in 3 week-old pups following challenge with 8.3xLD50 of tetanus toxin. The survival rates for the pups born to Alum-IM, MNP, ID and IM mothers were 100%, 100%, 87.5% and 13%, respectively (Fig. 6A) and correlated well with the neutralizing antibodies (0.74) (Suppl. Fig. 1E). A titer of 5 found in offspring from ID immunized mothers was sufficient to confer 87.5% protection against the toxin (Fig. 6B). The correlation between binding antibodies and neutralizing titers was lower in the neonates than in the mothers (R2 = 0.94 vs. 0.99 respectively). Six week old pups from Alum-IM and MNP-immunized mothers were fully protected against lethal infection whereas the offspring from ID and IM immunized mothers showed 20% and 0% survival rates respectively (Fig. 6C). Offspring challenged with the same dose of tetanus toxin 12 weeks after birth did not survive the infection although the pups from MNP immunized mothers delayed developing symptoms of disease until day 4, suggesting a limited degree of protection (Fig. 6D). Statistical analysis of survival rates showed significant differences between MNP and IM vaccination up to week 6 (pweek3 b 0.0001, pweek6 = 0.0003). While there was not a significant difference between MN and ID vaccination at week 3, the former outperformed intradermal vaccination at week 6 (p = 0.008) and week 12 (p = 0.03).
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The data from this experiment confirm the superiority of MNP vaccination, since the passive immunity observed in the offspring of vaccinated mothers was robust and long-lived, with antibodies fully protective at least up to 6 weeks after birth. 4. Discussion In this study, we examined the host immune responses to TT induced by skin delivery and compared them to conventional intramuscular immunization with TT and to commercially available Alum® adjuvanted vaccine administered IM. We developed MNPs encapsulating TT without adjuvant for cutaneous vaccination of pregnant and non-pregnant BALB/c mice and found that a single dose of 5 Lf (flocculation units) delivered with MN was sufficient to induce robust humoral immune responses sufficient to successfully protect the vaccinated animals against a lethal challenge dose with tetanus toxin (50xLD50). Matsuo et al. reported that cutaneous immunization of non-pregnant rats with dissolving MNPs containing unadjuvanted TT induced effective immune responses as well as IM and ID routes of TT delivery after 5 vaccine doses [55]. Our delivery system demonstrated superiority to IM and ID routes in both non-pregnant and pregnant cohorts and their offspring. Immunization with MNPs improved affinity maturation as seen by isotype class switching when compared to ID or IM routes. It also conferred a greater passive immune response amongst offspring as well as significant protection against high challenge dose with tetanus toxin compared to the offspring of mice vaccinated intramuscularly during pregnancy. Insertion of MNPs in the skin causes micro-injury of keratinocytes, releasing cytokines involved in innate cell recruitment in the area of needle insertion and antigen delivery [56]. Thus skin vaccination can exert an adjuvant effect per se potentially replacing the need for an adjuvant necessary to increase the immunogenicity of toxoid. Our findings agree with this hypothesis but only for the non-pregnant population in which MNP administration of TT without adjuvant induced levels of total immunoglobulins similar to IM vaccination of TT with Alum as
Fig. 5. Passive immunity in offspring of vaccinated with tetanus toxoid mice. Passively transferred TT-specific antibodies were determined in serum of offspring from vaccinated mice with ELISA. (A) Total IgG (B) IgG1 (C) IgG2a. Values are expressed as mean ± SEM (n = 5–12 mice per group. (D) IgG1 vs IgG2a antibody ratio was calculated for weeks 3 and 4. Alum IM: offspring of mothers immunized IM with Alum-TT vaccine (n = 5); MN: offspring of mothers immunized with MNPs (n = 10); ID: offspring of mothers immunized intradermally (n = 10); IM: offspring of mothers immunized intramuscularly (n = 10); N: naïve (n = 5).
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Fig. 6. Protective immune responses in offspring of vaccinated mothers. Independent cohorts of offspring born from mothers immunized with 5Lf Tetanus Toxoid using MNPs (MN) (n = 10), intradermal (ID) (n = 10), or intramuscular (IM) injection (n = 10) or immunized IM with Alum-TT vaccine (n = 5) as well as naïve (N) controls (n = 5) were challenged with 8.33xLD50 tetanus toxin at (A) 3-weeks, (C) 6-weeks, and (D) 12-weeks of age and followed up to 4 days for survival. (D) Neutralizing TT-specific antibody titers were assessed in pooled-serum of 3-week old pups. The titer was the highest dilution of pooled serum in PBS that neutralized 2xLD50 Tetanus Toxin. Abbreviations are as listed in the legend for Fig. 5.
adjuvant. Interestingly, although the IM administered Alum® TT vaccine induced almost 2-fold higher IgG responses than the MNPs administered unadjuvanted TT in pregnant mice, the immunoglobulin avidity and affinity were similar between these vaccination routes in both nonpregnant and non-pregnant cohorts and both groups survived challenge with tetanus toxin. The underlying mechanisms need to be further investigated. The potency of passively transferred antibodies in the newborns reflected to a certain extent the differences seen in antibody titers in the mothers. The most significant correlation was identified between neutralizing antibody titers and percent survival in 3 week old newborns. Interestingly, mice from MNP-vaccinated mothers had significantly higher IgG2a titers than neonates from Alum® TT vaccinated mothers whereas these titers were not different in the mothers. These data suggest that this enhanced response may be partly attributed to passively transferred maternal antibodies and partly to the developing immune system of the offspring. They also agree with a recent report from our group showing that young mice vaccinated with influenzacoated metal MNPs had improved IgG2a responses compared to IM vaccinated animals [57]. Two types of effector CD4+ T helper cell responses can be induced by professional antigen presenting cells, designated Th1 and Th2, each designed to eliminate different types of pathogens. Th2 responses are critical for humoral immunity necessary for control of bacterial pathogens (i.e. tetanus) due to neutralizing antibody production by IL-4-activated B cells. While the responses in immunized non-pregnant cohorts were primarily of the Th2 type, as expected by the nature of TT [58], the Th2 bias was significantly less in the pregnant cohort as determined by increased IgG2a production reflective of Th1 type, mainly after MNP vaccination. No changes were observed in the IgG1/IgG2a ratios seen
in Alum-IM vaccinated pregnant and non-pregnant cohorts that demonstrated a clear Th2 bias. It is worth noting that despite the 3-fold difference of TT neutralizing antibody titers between MNP (GMT 80) and ID immunized (GMT 10) non-pregnant mice, both groups fully survived a high lethal challenge whereas neither the MNP (GMT 20) nor the ID (GMT 10) immunized pregnant mice showed full protection against the same dose. These data suggest that antibody quality, which plays a very important role in protective immunity, may be affected at transcriptional or translational levels by pregnancy hormones. Although further product development and human clinical studies are needed, the results of this study demonstrate the potential of MNP vaccination to protect pregnant women and their newborn babies against tetanus. Our aim is to achieve a protective immune response with reduced vaccine doses and elimination of possible reactogenicity of aluminum salts by harnessing the immunological power of skin immunization. 5. Conclusions The first aim of the study was to assess the immune responses to TT elicited in pregnant and non-pregnant mice after intramuscular or cutaneous vaccination with either MNPs or intradermal injection. It was shown that unadjuvanted TT could be encapsulated into MNPs and delivered to murine skin. MNP-vaccination with unadjuvanted TT induced a significantly more robust immune response as well as superior protection to tetanus toxin challenge compared to intramuscular vaccination. The antibody responses were comparable to commercially available Alum® adjuvanted vaccine administered intramuscularly. It was also observed that pregnant mice regardless of their immunization route
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had lower humoral immune responses and were less protected against lethal tetanus toxin challenge than their non-pregnant controls. The second aim of the study was to compare the passive immune responses in the offspring of immunized pregnant mice. The passive immunity was more robust and longer lasting in the offspring of MNPvaccinated mothers. The offspring of Alum® adjuvanted vaccine and MNP vaccinated mothers were conferred complete protection against lethal tetanus toxin challenge at six weeks of age while the offspring of intramuscularly vaccinated mothers without Alum® had no protection. These results in the mouse model have demonstrated the potential of MNP technology to reduce the incidences of maternal and neonatal tetanus using a simple, patch-based delivery system.
[11]
[12]
[13]
[14] [15]
[16]
Research funding The work was supported by United States Agency for International Development (AID-OAA-F-13-00083 award). Conflict of interest Mark Prausnitz is an inventor of patents that have been licensed to companies developing microneedle-based products, is a paid advisor to companies developing microneedle-based products, and is a founder/shareholder of companies developing microneedle-based products (Micron Biomedical). The terms of this arrangement have been reviewed and approved by Georgia Tech and Emory University in accordance with their conflict of interest policies. E. Stein Esser, Richard Compans and Ioanna Skountzou have filed invention disclosure for tetanus skin immunization. Elena V. Vassilieva and Andrey A. Romanyuk, declare that they have no conflict of interest.
[17] [18] [19] [20]
[21]
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Acknowledgements We thank Dahnide Taylor-Williams, Erin-Joi McNeal, Nadia Lelutiu and Donna Bondy for their valuable technical and administrative support. We thank Haripriya Kaluri for her assistance to obtain unadjuvanted tetanus toxoid. We thank Serum Institute of India for providing tetanus toxoid monobulk.
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Appendix A. Supplementary data [30]
Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.jconrel.2016.06.026. [31]
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