The safety profile of a recently described novel archaeal lipid mucosal vaccine adjuvant and delivery (AMVAD) system capable of eliciting robust antigen-specific mucosal and systemic immune responses was evaluated in female Balb/c mice (10/group) using ovalbumin (OVA) antigen. Mice were intranasally immunized (0, 7, and 21 days) with a vaccine comprising 1 μg OVA (0.05 mg/kg body weight) formulated in 0.04 mg total polar lipids extract (2.17 mg/kg body weight) of Methanobrevibacter smithii constituting the AMVAD system. Control groups were similarly immunized with 10-fold higher AMVAD vaccine dose (0.54 mg OVA and 21.7 mg lipid per kg), saline, 10 μg OVA in saline, or 0.04 or 0.4 mg lipid constituting empty AMVAD (no OVA) in saline, or were naïve mice. Clinical signs, rectal temperature, and body weight were monitored once daily or as appropriate. Half the mice in each group were euthanized at 2 days after the first immunization. Blood was collected for clinical chemistry analyses. Major organs (heart, lungs, kidneys, liver, spleen, thymus, and brain) were examined macroscopically and histologically. The remaining mice were euthanized at 29 days and blood and organs collected for analyses as done at 2 days. Feces collected at 27 days, and sera, bile, and nasal lavage at 29 days, were assayed for antibody responses. Based on clinical symptoms, temperature, body weight changes, serum clinical chemistry, and tissue histopathology, there were no overt toxicities associated with OVA/AMVAD or empty AMVAD vaccines. There were no antibodies elicited against the lipids comprising the AMVAD system. These results demonstrate that at 10-fold excess dose of that required for vaccine efficacy, intranasally administered AMVAD vaccine appears to be relatively safe.
To better protect the host from diseases caused by pathogens that invade via mucosal surfaces such as the respiratory, gastrointestinal, and urogenital tracts, there are ongoing efforts to develop safe and efficient mucosal adjuvants and vaccine delivery systems (Holmgren et al. 2003; Yuki and Kiyono 2003; Neutra and Kozlowski 2006). Most vaccines are currently administered via systemic routes, but these are usually not efficient in generating protective mucosal immunity (Mestecky et al. 1997; Singh and O’Hagan 2002) that would help prevent pathogen attachment, replication, and invasion (Holmgren et al. 2003). The relatively few mucosal vaccines on market today are mostly based on attenuated pathogens or killed whole cells (Holmgren et al. 2003; Neutra and Kozlowski 2006). Irrespective of their efficacy, approaches based on attenuated pathogens, dead cells, live vectors, and on nonreplicating adjuvants/delivery systems such as cholera toxin (CT), Escherichii coli heat-labile enterotoxin (LT), immune stimulating complexes (ISCOMs), surfactants, and DNA, have real or perceived safety concerns (Lemoine, Francotte, and Preat 1998; Ogra, Faden, and Welliver 2001; O’Hagan and Rappuoli 2004; Neutra and Kozlowski 2006). Because the more defined, acellular or subunit antigens are usually not highly immunogenic on their own (Ogra, Faden, and Welliver 2001; Ryan, Daly, and Mills 2001), mucosal adjuvants and delivery systems would especially be useful for developing vaccines based on such antigens. Hence, there is continued interest in developing safe mucosal adjuvants and vaccine delivery systems (Levine 2003; Holmgren and Czerkinsky 2005).
It is generally recognized that the nasal route is less harsh than the oral route for vaccine delivery, and it requires comparatively less adjuvant and antigen dose for efficacy (Holmgren et al. 2003; Yuki and Kiyono 2003). In addition to eliciting mucosal immune responses at the local respiratory and distal mucosal (e.g., genital and intestinal) sites, intranasal (i.n.) immunization can also promote systemic immune responses (Lemoine, Francotte, and Preat 1998; Ogra, Faden, and Welliver 2001; Neutra and Kozlowski 2006). Thus, this route of immunization would be especially useful for protection against respiratory pathogens.
We recently described a novel, nonreplicating, particulate, archaeal polar lipid-based mucosal adjuvant and vaccine delivery (AMVAD) system (Patel et al. 2007; Patel et al. 2008). Intranasal immunization of ovalbumin (OVA) adjuvanted with the AMVAD system, but not with archaeosomes (liposomes made from archaeal polar lipids), elicited robust, sustained, memory-boostable, OVA-specific mucosal and systemic immune responses in mice (Patel et al. 2007). The AMVAD vaccines were prepared by the interaction of small unilamellar archaeosomes, OVA and CaCl2 (Patel et al. 2007; Patel et al. 2008). Unlike archaeosomes, which consist of small (average diameters of ca. 100 nm), individual, nonaggregated spherical vesicles, the AMVAD structures are composed of much larger spherical structures that have aggregated together like a bunch of grapes (Patel et al. 2008). However, both the archaeosomes (Choquet et al. 1994) and AMVAD structures contain aqueous capture volume or compartments (Patel et al. 2008). Although the safety profile of archaeosomes administered to mice via systemic routes (subcutaneous, intravenous) indicate that archaeosomes and archaeal polar lipids are relatively safe (Patel et al. 2002; Omri, Agnew, and Patel 2003), there is no information on the safety profile of archaeosomes or AMVAD vaccines administered via the i.n. route. However, the safety, and efficacy, of a vaccine/adjuvant is influenced by the administration route used (Byrd and Cassels 2006; Aguilar and Rodriguez 2007). For example, compared to the systemic route, i.n. vaccination needs to keep in perspective the potential for adverse reactions at the nasal/respiratory surfaces and in the central nervous system (Fujihashi et al. 2002; Levine 2003; Yuki and Kiyono 2003; Byrd and Cassels 2006). Therefore, the objective of the current study was to evaluate the safety profile of the novel AMVAD vaccines upon i.n. immunization.
MATERIALS AND METHODS
Materials
The total polar lipid (TPL) extract was obtained from the archaeal species Methanobrevibacter smithii (DSM 2375). The total lipid extract (TLE) of M. smithii grown in a 75-L fermenter vessel was obtained by chloroform/methanol/water extraction of the biomass, and the TPL extract was obtained by cold acetone precipitation from the TLE, as described previously (Patel et al. 2007). The TPL extract was stored in chloroform at 4°C.
Animals
Specific pathogen–free, female, Balb/c mice were purchased from Charles River Laboratories (Montreal, P.Q., Canada), and entered the experiments at 8 to12 weeks of age. The mice were housed and used as per the Canadian Council on Animal Care Guide to the Care and Use of Experimental Animals. All animal care/use protocols were approved by the Institutional Animal Care Committee.
Preparation and Characterization of OVA/AMVAD and Other Vaccine Formulations
Ovalbumin was formulated with the AMVAD system to obtain the OVA/AMVAD vaccine as described earlier for making the OVA/AMVAD-ANW (ANW-admixed not washed) formulation (Patel et al. 2008), except for the indicated changes below. Briefly, the vaccine was prepared by admixing empty archaeosomes with OVA, and then adding CaCl2 to covert to OVA/AMVAD. The starting ratio (w/w) of TPL:OVA was changed to 40:1 (from 22.2:1) and a final step for AMVAD size reduction was included. In the current work, this vaccine formulation is referred to as OVA/AMVAD, indicating that OVA was adjuvanted with AMVAD system prepared from the TPL extract from M. smithii. Briefly, 20 mg of a thin, dried film of TPL was hydrated in 0.9 ml of sterile Milli-Q water, followed by brief period of bath sonication (Model FSH-60H, 130 W, 40 kHz; Fisher Scientific, Ottawa, Ontario, Canada) to obtain small, empty, unilamellar archaeosomes. The average diameter of the archaeosomes was ca. 100 nm, as determined by number-weighted gaussian size distribution (Model 350 Nicomp particle size analyzer; Nicomp, Santa Barbara, CA, USA). The archaeosome suspension was supplemented with 0.1 ml of a filter sterilized OVA stock solution to add 0.50 mg OVA (lipid:OVA ratio of 40:1; 0.4 mg TPL:10 μg OVA). The suspension was briefly mixed by vortexing, and converted into OVA/AMVAD formulation by dropwise addition of an autoclaved (121°C, 20 min) 0.1 M CaCl2 solution while vigorously mixing the suspension by vortexing. The conversion of archaeosomes into AMVAD structures was confirmed by phase-contrast microscopy observation (ca. 1250× magnification; Olympus Model BX51 TF microscope; Olympus America, Melville, NY, USA), as described earlier (Patel et al. 2007, 2008), which indicated the absence of the small, individual, spherical archaeosome vesicles (barely visible at this magnification) and the presence of larger, aggregated, spherical structures exhibiting phase-bright perimeters and some phase-bright areas within the aggregates. Five sterile glass beads (3-mm diameter) were added to the formulation in a test tube and the tube was subjected to 3 to 5 min of sonication in a bath sonicator, to reduce the size of the bulk of the AMVAD structures to <5 μm width (in the range of 1 to 5 μm). The formulation was not washed to remove any free, soluble OVA that was not associated with the AMVAD particulate structures, but this amount was determined by centrifuging an aliquot of the formulation (16,000 × g, 10 min) to sediment the particulate AMVAD structures, and determining the quantity of OVA present in the supernatant using a modified Lowry colorimetric protein assay. The average width of the AMVAD structures in the formulation was determined by randomly measuring the widths of a minimum of 200 AMVAD structures from images of the formulation that were captured under phase-contrast microscopy with a digital camera (Micropublisher 5.0 RTV; QImaging, Burnaby, BC, Canada), using the QCapture Pro software (QImaging).
An empty AMVAD formulation (adjuvant/delivery system alone, no OVA) was prepared as described for OVA/AMVAD above, except that the TPL was hydrated in 1 ml of water because there was no supplementation with OVA.
A control vaccine consisting of OVA/0.85% NaCl (OVA/saline) was prepared by filter sterilizing (0.22-μm filter) a 0.2 mg/ml OVA stock solution in 0.85% saline.
All vaccine formulations were prepared aseptically using pyrogen-free glassware and sterile MilliQ water, and were stored at 4°C until use. Just prior to immunization, an aliquot of the empty AMVAD or the OVA/AMVAD formulation was diluted to the immunization dose in a final concentration of 0.85% saline/15 mM CaCl2 (pH 7.1).
Experimental Design
Groups of mice (n = 10) were intranasally immunized with OVA-AMVAD-L (1 μg OVA/0.04 mg lipid) or 10-fold higher OVA/AMVAD vaccine (10 μg OVA/0.4 mg lipid), or with empty AMVAD, OVA/saline, or saline-control vaccine (Table 1) at 0, 7, and 21 days. For the i.n. immunization (50-μl volume), the mice were anesthetized by intraperitoneal (i.p.) injection of ketamine and xylazine at 0.1 and 0.05 mg/g body weight, respectively, in 0.25 ml injectable saline. A naïve-control group was also included. Mice were observed at least once daily for the first 2 days after each immunization (or longer as needed), and at weekly intervals thereafter, for clinical signs such as hyperactivity, lethargy/sedation, aggressive behaviour, piloerection (ruffled or erect fur), alopecia, dehydration, salivation, nasal discharge, and diarrhea. The overall clinical symptoms for each mouse were scored on a sliding scale of 0 to 4, where 0 represented the normal, active, healthy mouse and 4 the moribund mouse. The body weight of mice was monitored before immunizations at 0, 7, and 21 days, at up to 2 days post each immunization, and at days 14 and 29. The change in body weight was expressed as percent change in body weight from that at 0 day. The body temperature was taken using an anal probe (same time each day, before handling the mice for anything else), at similar time points to the body weight.
At day 2 (i.e., 48 h after the first immunization) and day 29 (i.e., 7 days after the last immunization), five mice from each group were euthanized by CO2 asphyxiation. Blood samples were collected by cardiac puncture and sera were separated and stored at − 20°C till used for clinical blood chemistry analysis. Spleen, lungs, liver, heart, kidneys, and thymus from each mouse were collected, observed macroscopically, and weighed. The organ weights were normalized by expressing them as percent relative organ weights (organ weight as a percentage of the body weight) to compensate for any differences solely due to the differences in the respective body weights of mice. These organs, and the brain, were fixed in 10% neutral-buffered formalin (pH 7.0). The organs were sectioned and hematoxylin-stained (Department of Pathology and Laboratory Medicine, University of Ottawa, Ottawa, Ontario, Canada) for histopathology examination. In addition, fecal samples, and nasal wash and bile samples were collected at days 27 and 29, respectively, processed, and stored as described previously (Patel et al. 2007), for determination of anti-OVA immunoglobulin A (IgA) and anti-lipid IgA immune responses.
Measurement of OVA-Specific IgA and IgG Responses by ELISA
For establishing the efficacy of the OVA/AMVAD vaccines in eliciting of OVA-specific mucosal and systemic immune responses, indirect enzyme-linked immunosorbent assays (ELISA) were used for the determination of OVA-specific IgA antibodies in 1:2 diluted fecal samples collected at 27 days, or in 1:2 diluted nasal wash, 1:20 diluted serum samples collected at 29 days, and OVA-specific IgG antibodies in 1:2000 diluted serum samples collected at 29 days. Briefly, 96-well flat-bottom Immunolon 2 microplates (Thermo Electron, Milford, MA, USA) were coated (4°C overnight) with 5 μg OVA/well in 100 μl of 0.1 M bicarbonate buffer (pH 9.6), the plates were washed twice, and then blocked (1 h, room temperature) with 5% bovine serum in phosphate-buffered saline (PBS). Aliquots of appropriately diluted samples (100 μl/well) were added to duplicate wells, and the plates were incubated at room temperature for 3 h. After washing the plates 3×, alkaline phosphatase–conjugated goat anti-mouse IgA (1:1000) or IgG (1:3000) were added (all from Caltag Laboratories, Burlingame, CA, USA), plates incubated for 1 h (room temperature), and color reactions were developed by the addition of p-nitrophenyl phosphate substrates (Kirkegaard and Perry Laboratories, Gaithersburg, MD, USA). The optical density (OD) was measured at 405 nm after 10- to 60-min incubation periods, using an automated ELISA plate reader (Model 354;Thermo Labsystems, Helsinki, Finland) and Multiskan Accent software (Thermo Labsystems).
Measurement of Lipid-Specific IgA and IgG Responses by ELISA
The potential for eliciting antibodies against total polar lipids (anti-TPL or anti-lipid antibodies) comprising the AMVAD vaccine was assessed by ELISA assay as described above, except for the changes indicated below. Each well of the plate was coated with 1 μg of TPL of M. smithii in the form of empty, unilamellar (average diameters of ca. 100 nm) archaeosomes, instead of OVA. Preliminary assays using different amounts of archaeosome lipid coating and positive mouse sera obtained from previous studies where mice had been subcutaneously immunized with empty archaeosomes established that this level of coating was optimal. For assay of anti-lipid IgA antibody responses, the fecal and nasal wash samples were used without dilution and serum and bile samples were diluted 1:10. For assay of anti-lipid IgG, IgG1, and IgG2a antibody responses, the serum samples were diluted 1:50. The alkaline phosphatase–conjugated goat anti-mouse IgA (1:1000), IgG1 or IgG2a (1:2000), or IgG (1:3000) used were from Caltag Laboratories. Color reaction developed after a 60-min incubation subsequent to the addition of p-nitrophenyl phosphate substrate was read as OD at 405 nm.
Clinical Blood Chemistry
The sera were assayed for the levels of alkaline phosphatase (ALP), serum glutamic pyruvic transferase (SGPT), glucose, blood urea nitrogen (BUN), creatinine, bilirubin, albumin, globulin, and total protein using the Roche Hitachi 917 Analyzer (Vita-Tech, Markham, Ontario, Canada).
Statistical Analyses
All data are reported as mean ± standard deviation (SD) for each group. Differences between treatment groups were assessed by two-tailed Student’s t test or one-way analysis of variance (ANOVA) followed by Dunnet’s test, as appropriate. Differences were considered significant at p < .05.
RESULTS
The ratio (w/w) of lipid:Ca2+ in the OVA/AMVAD and empty AMVAD formulations was 25:1, and the final CaCl2 concentration in these formulations was 14.9 mM (Table 1). Approximately 95% and 75% of AMVAD structures in the OVA/AMVAD and empty AMVAD formulations were of <5-μm average width, respectively. Less than 1% and 6% of the AMVAD structures in OVA/AMVAD and empty AMVAD formulations, respectively, were >10 μm wide. Of the total OVA in the OVA/AMVAD formulation (10 μg OVA/0.4 mg TPL), ca. 60% was present in the free, soluble form, the rest being associated with the AMVAD particles.
The OVA immunization dose of 1 or 10 μg (Table 1) in AMVAD vaccines was based on the total OVA in the formulation, with the lipid:OVA ratio (w/w) being kept constant at 40:1. The anti-OVA IgA antibody responses measured in the sera, fecal, and nasal wash samples of the group immunized with the OVA/AMVAD-L (1 μg OVA/0.04 mg lipid) vaccine were comparable to the corresponding responses in the group immunized with the OVA/AMVAD (10 μg OVA/0.4 mg lipid) vaccine (Figure 1), which had a 10-fold higher antigen and AMVAD lipid immunization dose. The anti-OVA serum IgG antibody response was significantly higher (p < .05) with the higher vaccine dose (Fig. 1). As expected and also seen in our earlier studies (Patel et al. 2007, 2008), the saline, OVA/saline, or empty AMVAD vaccines elicited little to no OVA-specific mucosal immune responses (data not shown). These results demonstrate that an AMVAD vaccine dose as low as 1 μg OVA/0.04 mg lipid gave robust mucosal and systemic immune responses that were generally comparable to those obtained with a 10-fold higher immunization dose (10 μg OVA/0.4 mg lipid). Therefore, the safety of the OVA/AMVAD vaccines was evaluated at these two doses.
To determine if antibody responses were elicited against the lipid adjuvant/carrier itself comprising the AMVAD formulation, anti-TPL (anti-lipid) antibody responses were determined by ELISA. Assay for anti-TPL IgA antibodies in feces collected at 27 days, and in nasal wash samples collected at 29 days, indicated that empty AMVAD or OVA/AMVAD vaccines, at the respective low and 10-fold higher doses, did not elicit anti-TPL IgA antibodies as compared with the OVA/saline-immunized control group (Figure 2; p > .05), even after 60 min of color development and using samples that were undiluted or less diluted than those used for measuring anti-OVA antibody responses. The responses were also negligible and similar in the naïve and saline-vaccinated groups (Figure 2), indicating that the responses measured were baseline responses. The anti-lipid IgA responses in the sera collected at 29 days were below detectable levels (data not shown). Similarly, the anti-TPL IgA antibody responses (at 29 days) in the bile and the anti-TPL IgG1, IgG2a responses in the 29-day sera of mice immunized with the OVA/AMVAD or empty AMVAD vaccines (at the lower or higher dose of each) were negligible, and comparable to those seen with the OVA/saline-vaccinated group (data not shown). The anti-TPL IgG antibody responses in the OVA/AMVAD-and empty AMVAD-immunized groups were also very low and comparable to those seen in the OVA/saline-control group (Figure 2; p > .05).
None of the mice in the experiment died due to the treatment or had to be euthanized due to sickness. None of the mice in any group exhibited overt symptoms such as hyperactivity, lethargy/sedation, aggressive behavior, alopecia, dehydration, salivation, nasal discharge, or diarrhea. The clinical scores of mice in each group were between rating of 0 and 1, as indicated in Table 2. At 1 day post each immunization at 0, 7, and 21 days, the groups immunized with empty AMVAD or OVA/AMVAD had slightly poorer clinical scores of 1, especially at the higher lipid doses, but these scores were not significantly different from the average clinical score of 0 (normal, healthy, active) for the OVA/saline-control group. However, by second day post each immunization, the scores in these groups had recovered to levels similar to the naïve, saline-vaccinated, and OVA/saline-vaccinated groups. Thus there do not seem to be dose-related adverse clinical symptoms associated with the empty AMVAD or OVA/AMVAD vaccines.
The average body temperature of all mice at 0 day (before any treatment) was 38.35°C (range form 38.11°C to 38.54°C). The temperature of all mice in the different groups was between 37.64°C and 38.74°C during the observation period, and there were no apparent trends (up or down) in the change of body temperature in any group of mice to suggest treatment related effects. Essentially, the small variations in temperature were random and generally similar between all groups, including the naïve, saline-immunized, and OVA/saline-immunized groups.
The average body weight of all mice at 0 day was 18.43 ± 1.0 g. At 1 day (24 h post first immunization), all groups of mice, including the naïve, lost an average of 0.3% (naïve group) to 5.2% of the prechallenge body weight (Figure 3). Compared with the loss of 2.4% in the OVA/saline group, the loss in the empty AMVAD (0.4 mg dose) and OVA/AMVAD (10 μg OVA/0.4 mg lipid) groups was significantly higher, p < .05 and .01, respectively. By 7 days, all groups had regained the original weight, except for the groups immunized with the empty AMVAD-L (lower AMVAD lipid alone dose) and the OVA/AMVAD (10-fold higher OVA/AMVAD lipid dose) vaccines. The changes in the body weight at any of the other indicated time points beyond 8 days (Figure 3) were not significantly different (p > .05) compared with the OVA/saline group. At 29 days, all groups had gained weight, from 4.3% in the OVA/saline group to 3.0% in the OVA/AMVAD-vaccinated group. These results indicate some minor, transient adverse body weight change effects at the 10-fold higher OVA/AMVAD vaccine dose, but the mice recover to gain body weight comparable to the OVA/saline-immunized group. No adverse effects were noted with the lower OVA/AMVAD-L, or the higher or lower empty-AMVAD vaccine doses.
The gross necropsy of all major organs (spleen, lungs, liver, thymus, and heart) was normal, and comparable to that of organs in the naïve and the OVA/saline-immunized group.
The relative organ weights of spleen, lungs, and liver, for each treatment group after 2 and 29 days, are indicated in Figure 4. Compared with the OVA/saline-vaccinated group, there were no significant differences (p > .05) in the corresponding percent relative organ weights from groups immunized with empty AMVAD, empty AMVAD-L, OVA/AMVAD, OVA/AMVAD-L, or saline, or from the naïve group at 2 and 29 days, respectively. Similarly, there were no differences in the percent relative weights of thymus, heart, or the kidneys at 2 or 29 days, respectively (data not shown).
Histopathologically, there was no remarkable change in any tissues from the saline-immunized, OVA/saline-immunized, or the naïve groups. At the day 2 analyses, the lungs from the groups immunized with empty AMVAD-L, empty AMVAD, OVA/AMVAD-L, and OVA/AMVAD showed various degrees of inflammatory responses. The lungs from the higher dose of empty AMVAD (0.4 mg lipid) and OVA/AMVAD (10 μg OVA/0.4 mg lipid) were most remarkable and those from the corresponding 10-fold lower doses were less severe. Overall, the lungs from these groups of mice killed at day 2 showed mild to moderate infiltration of admixed neutrophils and mononuclear cells in the areas adjacent to airways and sometimes in the airway lumens. However, these acute inflammatory changes had completely subsided and resolved by day 29. By this time, the lungs showed the presence of multiple mononuclear cell aggregates, of small to medium size, in the peribronchial and perivascular areas. There was no remarkable change in the brain, thymus, heart, spleen, liver, or kidneys of these mice at either time point.
The alkaline phosphates (ALP), serum glutamic pyruvic transaminase (SGPT), glucose, blood urea nitrogen (BUN), creatinine, and bilirubin in sera obtained at 2 and 29 days, respectively, from all treatment groups are shown in Figure 5. Compared with the OVA/saline group, significant differences (p < .05) in the other treatment groups in the corresponding serum parameter measured at 2 or 29 days respectively, were as indicated in Figure 5. It can be seen that there were no significant dose-related differences associated with the OVA/AMVAD vaccinations. Similarly, there were no significant treatment-related differences (p > .05) in the total protein, albumin, globulin, and the ratios of albumin/globulin between OVA/AMVAD-vaccinated groups, the saline group, and the naïve group, as compared with the OVA/saline-control group (data not shown). Therefore, there are no overall OVA/AMVAD vaccine dose-related adverse effects on hepatic (such as SGPT, ALP, globulin, glucose, bilirubin, albumin, and total protein) or renal (such as BUN and creatinine) function.
DISCUSSION
Although it has been previously shown that TPL extract of different archaeal species, including from M. smithii, are relatively safe upon intravenous, oral, and subcutaneous administration to mice in the form of archaeosomes (Omri, Agnew, and Patel 2003), or archaeosome-adjuvanted vaccines administered subcutaneously (Patel et al. 2002), the safety of these archaeosomes or of AMVAD system has not been evaluated upon i.n. administration. It is acknowledged that the safety as well as the efficacy of a vaccine/adjuvant can be impacted by the selected route of immunization (Byrd and Cassels 2006; Aguilar and Rodriguez 2007). Additionally, whereas unilamellar archaeosomes used in previous studies consisted of small (average diameters of 100 nm), individual, nonaggregated vesicles, the AMVAD structure is distinct in that it comprises of much larger spherical structures that have aggregated together like bunches of grapes (Patel et al. 2008). In the current study, ca. 95% and 75% of the AMVAD structures in OVA/AMVAD and empty AMVAD vaccines, respectively, were less than 5 μm in width (in the range of 1 to 5 μm). For the above reasons, it was essential to evaluate the detailed safety profile of AMVAD vaccines administered by the i.n. route.
We recently demonstrated that i.n. immunization of mice with an OVA/AMVAD vaccine, prepared from the TPL extract of M. smithii using a standard preparation protocol (Patel et al. 2007), elicited robust, sustained, and memory-boostable mucosal and systemic OVA-specific antibody responses and CD8+ cytotoxic T-lymphocyte responses. Subsequently, we demonstrated that OVA/AMVAD formulations prepared by a simpler admixing protocol also elicited robust OVA-specific immune responses (Patel et al. 2008). The admixing protocol avoids centrifugation and washing steps to remove free, soluble antigen in the formulation, as well as the need for quantization of antigen specifically associated with the AMVAD particulate structures, because the immunization is based on the total amount of antigen in the formulation. The admixing protocol results in consistent formulations from batch to batch, including the percentage of the total OVA antigen associated with the AMVAD particulate structures. The current study confirmed earlier observations (Patel et al. 2007, 2008) that little to no anti-OVA IgA antibody responses were observed in sera, nasal wash, vaginal wash, or bile samples from mice immunized with OVA/saline or empty AMVAD vaccines. The current study further demonstrated that the mucosal and systemic immune responses obtained in mice at an immunization dose of 1 μg OVA/0.04 mg lipid (OVA/AMVAD-L vaccine) were generally comparable to those attained with a 10-fold higher vaccine dose (OVA/AMVAD vaccine; 10 μg OVA/0.4 mg lipid; 0.54 mg OVA/21.7 mg lipid/kg body weight). The OVA immunization doses were based on the total OVA in the formulations, both AMVAD particulate structure associated and the free, soluble OVA. Therefore, the safety profile of the AMVAD vaccine in this study was evaluated by including a vaccine dose that was at least 10-fold higher (10 μg OVA/0.4 mg lipid) than the minimum currently known to be required (1 μg OVA/0.04 mg lipid) for eliciting robust mucosal immune responses in the mouse model. Control groups were immunized with comparable amount (lipid weight basis) of empty AMVAD vaccines, saline only, 10 μg OVA/saline vaccine, or were naïve mice.
The elicitation of antibody responses against the adjuvant or carrier/delivery vehicle component of a vaccine can suppress the immune responses raised against the antigen, especially upon repeated use of the adjuvant/carrier to immunize the host (Mestecky et al. 1997; Haneberg, Herland Berstad, and Holst 2001). Preexisting immunity against the mucosal adjuvant cholera toxin B (CTB) has been reported to inhibit the mucosal and serum antibody responses elicited against a dextran antigen upon subsequent i.n. immunization with a dextran-CTB conjugate vaccine (Bergquist, Lagergard, and Holmgren 1997). However, preexisting immunity against another mucosal adjuvant, LTK63, did not appear to affect its adjuvant efficacy upon i.n. vaccination (Vajdy and O’Hagan 2001). In this study, even with relatively undiluted samples and excessively long ELISA color development times of 60 min, little to no anti-lipid antibodies (IgA in feces, nasal wash, bile or sera; IgG, IgG1, IgG2a in sera) were detected upon i.n. vaccination with the higher (or lower) doses of empty AMVAD or OVA/AMVAD vaccines. An earlier study involving four subcutaneous immunizations of 1 mg TPL extract of M. smithii each, in the form of empty archaeosomes, had also showed absence of anti-lipid antibody responses (Omri, Agnew, and Patel 2003). The lack of antibody responses against the lipids constituting the AMVAD system suggests that there should be no anti-lipid antibody related concerns regarding the efficacy or safety of the AMVAD vaccines, upon repeated use.
There were no immunization-related severe toxicities or deaths in any of the groups of mice in this study. Further, there were no overt clinical signs observed in the vaccinated mice. Apart from transient observation of slightly ruffled fur, lasting about 1 day post some of the immunizations, with the 10-fold higher OVA/AMVAD (or empty AMVAD) vaccine dose, there were no other vaccination related observations in any groups of mice. In contrast, i.n. administration of 5 μg of an mLT– (a mutant form of heat-labile enterotoxin from Escherichia coli) adjuvanted vaccine in female Balb/c mice resulted in loss of activity, increase in huddling, and ruffled fur for up to 1 to 2 days post each immunization (Byrd and Cassels 2006). Although mice immunized with the higher OVA/AMVAD vaccine dose showed up to 7% loss of body weight, the loss was transient. The body weights of this group of mice subsequent to the 8th day of the experiment were not significantly different (p > .05) from that of the OVA/saline group, and by 29 days had gained weight similar to the other groups.
Macroscopic examinations of the major organs (liver, spleen, heart, lungs, and kidneys) showed no remarkable abnormalities. The relative organ weights in the groups vaccinated with OVA/AMVAD vaccine and empty AMVAD were not significantly different from the respective organs from the OVA/saline group. The detailed blood clinical chemistry also indicated that there were no adverse effects on the hepatic or renal function. As expected, histological examination of the lungs from the AMVAD-vaccinated groups showed acute recruitment of inflammatory cells (macrophages and neutrophils) into the airway at day 2, the inflammation being less at the lower AMVAD doses. Regardless, the lung inflammation in these groups of mice had subsided and was resolved by day 29. These inflammatory responses were probably essential for the recruitment and activation of antigen-presenting cells and other immune cells. By day 29, the lung of AMVAD-immunized mice showed the presence of large numbers of lymphoid aggregates in the peribronchial and perivascular areas. The presence of these lymphoid tissues is believed to be important for the induction of mucosal immune responses (Bienenstock and McDermott 2005). Apart from the immune and inflammatory responses seen in the lung, there was no remarkable change in the other tissues (liver, spleen, heart, thymus, kidneys, or brain) examined. The lack of any histological changes in the brain of AMVAD-immunized mice further indicated that there were no vaccine treatment-related adverse effect on this organ. In contrast to the safety concerns of i.n. administered toxin-based mucosal adjuvants such as LT (including some detoxified LT mutants) and CT regarding the potential to enter the central nervous system due to their ability to bind with GM1 gangliosides (Ryan, Daly, and Mills 2001; Fujihashi et al. 2002; Levine 2003; Yuki and Kiyono 2003), histological examination of the brain tissues from the groups of mice vaccinated with OVA/AMVAD and empty AMVAD vaccines suggests that the AMVAD system has no potential impact in the brain tissue.
Both CT and LT are potent mucosal adjuvants, but their use in humans is precluded due to their associated severe toxicities (Fujihashi et al. 2002; Yuki and Kiyono 2003; Holmgren and Czerkinsky 2005). Additionally, there are safety concerns related to the possible elicitation of immunity against “bystander” antigens (other antigens also present at the mucosal surface) with the use of these toxin adjuvants (Alpar et al. 2001; Ryan, Daly, and Mills 2001). Less toxic CT and LT mutants have been developed, but in general these resulted in diminished efficacy (Holmgren and Czerkinsky 2005). In the case of the AMVAD system, the antigen has to be closely associated with the particulate AMVAD structures for efficacy in eliciting antigen-specific immune responses. Intranasal immunization of mice with a vaccine prepared by simple admixing of preformed empty AMVAD structures with OVA failed to promote anti-OVA mucosal immune responses (G. B. Patel and W. Chen, unpublished observations). Therefore, the potential to generate bystander antigen immunity is less likely with the AMVAD system.
Although it is difficult to predict the safety or efficacy of a vaccine/adjuvant in humans, based on the evaluation in animal models (Holmgren and Czerkinsky 2005), such studies in mice are useful for leading up to evaluations in other animal species (Kenney et al. 2002) and eventually in humans. The detailed evaluation of AMVAD system in mice, at vaccine doses at least 10-fold higher than those required for eliciting strong antigen-specific mucosal and systemic immune responses, did not indicate any potential toxicity issues associated with i.n. vaccination. Based on the selected end points assessed, the AMVAD vaccines were generally well tolerated over the dose range evaluated.
