The cationic liposomal adjuvants CAF01 and CAF09 formulated with the major outer membrane protein elicit robust protection in mice against a Chlamydia muridarum respiratory challenge

Two cationic liposomal adjuvants CAF01 and CAF09 were formulated with the native or the recombinant Chlamydia muridarum major outer membrane protein (nMOMP and rMOMP). BALB/c mice were immunized with the four vaccine formulations using the subcutaneous followed by the intranasal (i.n.) routes. As positive controls mice were inoculated i.n. with live C. muridarum and negative controls received i.n. minimal essential medium (MEM). Four weeks after the last immunization mice were challenged i.n. with 10 4 inclusion forming units (IFU) of C. muridarum . Following the challenge the mice were weighed daily. At 10 days post-challenge the mice were euthanized, their lungs weighed and the number of C. muridarum IFU determined. Serum collected the day before the challenge showed that all four groups of mice immunized with CAF01, or CAF09 and MOMP had signiﬁcant C. muridarum -speciﬁc antibody titers. As determined by a T-cell lymphoproliferative assay, these four groups of mice also mounted robust cell mediated immune responses with high production of IFN-c and IL17 and low levels of IL-4. Following the challenge the four groups of mice lost signiﬁcantly less body weight than the MEM-immunized group. Lungs of mice vaccinated with CAF01, or CAF09, and nMOMP were signiﬁcantly lighter than those from mice immunized using rMOMP. The number of IFU recovered from the lungs of mice vaccinated with CAF01, or CAF09, and nMOMP was similar to the number of IFU recovered from mice immunized with live EB. Mice that received rMOMP had signiﬁcantly higher numbers of IFU than other groups. In conclusion, CAF01 and CAF09 elicited very robust protective humoral and cellular immune responses and were equally effective at adjuntavizing the C. muridarum MOMP. Mice vaccinated with nMOMP were signiﬁcantly better protected than those immunized with rMOMP, indicative of the importance of the structural conformation of this antigen in protection.


⇑ Corresponding author.
E-mail address: Imdelama@uci.edu(L.M. de la Maza).antibody levels and has also been demonstrated to cross prime CD8 T-cell responses [22].Several investigators have evaluated the efficacy of MOMP as a vaccine antigen, using various adjuvants, and observed different levels of protection against respiratory and genital challenges [13,14,18,[27][28][29][30][31].We formulated CAF01 and CAF09 with the native, or the recombinant, MOMP (nMOMP or rMOMP) with the goal of determining which of these adjuvant/ antigen combinations is the most effective at protecting mice against an intranasal (i.n.) challenge with C. muridarum.

Materials and methods
See supplemental material.

Humoral immune responses following vaccination
To determine the humoral immune responses in vaccinated mice, serum samples were collected the day before the i.n.challenge and C. muridarum-specific antibodies determined using EB as the antigen (Table 1).Positive controls immunized i.n. with live EB had an IgG geometric mean titer (GMT) of 51,200 (range: 51,200-51,200) while negative controls inoculated i.n. with MEM had a titer below the level of detection (<100).Animals vaccinated with CAF01 and nMOMP had similar IgG levels (GMT: 19,027; range 4000-64,000) to those immunized with CAF09 (GMT: 19,027; range 8000-32,000).Mice vaccinated using CAF01 (GMT: 6727; range 2000-32,000), or CAF09 (GMT: 9514: range 8000-18,000) and rMOMP also had similar IgG titers.
To determine what specific epitopes elicited antibody responses, serum samples were probed with 25 aa overlapping C. muridarum MOMP peptides (Fig. 1).Antibodies from controls immunized with live EB recognized peptides located almost exclusively to the four variable domains (VD) and constant domain (CD) 5 while no reactivity was obtained with sera from mice inoculated with MEM.Animals immunized with CAF01, or CAF09 and nMOMP, in comparison to those immunized using rMOMP, elicited broader repertoires of antibodies that included all VD and CD5.Mice vaccinated with CAF01 and rMOMP mounted antibody responses mainly to VD1 and CD5 while those immunized using CAF09 also had antibodies to VD3.
In conclusion, based on these findings, except in vaginal washes, no significant differences in antibody responses were observed between mice immunized with CAF01 versus CAF09.Broader and more robust antibody responses were observed in mice vaccinated with nMOMP versus rMOMP.

Cellular immune responses following vaccination
As a parameter of the C. muridarum-specific cellular immune responses, proliferation was determined using nylon-wool purified spleen T-cells (Table 2).The stimulation index (SI) of T-cells from mice vaccinated with live EB was 18.6 ± 2.3, while in animals inoculated with MEM the SI was 2.5 ± 0.2.Mice immunized with CAF01 and nMOMP or rMOMP had SI of 26.9 ± 2.7 and 30.6 ± 2.5, respectively, both significantly higher than the MEM control (P < 0.05).Similarly, animals vaccinated using CAF09 and nMOMP, or rMOMP, had SI of 40.5 ± 3.6 and 34.8 ± 6.1, respectively, both significantly higher than the MEM control (P < 0.05).
Mean IFN-c levels (pg/ml), as a measure of a Th1 response, were determined in supernatants from EB-stimulated T-cells (Table 2).Positive controls immunized with EB had high quantities (5470 ± 146) while those inoculated with MEM had low levels of IFN-c (76 ± 27).In mice vaccinated with CAF01 and nMOMP (3528 ± 945), or rMOMP (5293 ± 191), or with CAF09 and nMOMP (5416 ± 59), or rMOMP (4521 ± 690) levels of IFN-c were equivalent to those observed in controls immunized with EB.Mean levels of IL-4 (pg/ml), a marker of Th2 responses, were also present but at much lower levels ranging from (16 ± 7) in mice immunized with CAF01 and nMOMP and (26 ± 14) in animals receiving CAF09 and rMOMP, supportive of strong Th1 responses in all groups immunized with both CAF adjuvants.High levels of IL-17 were elicited in the four samples from mice immunized with CAF01 or CAF09.However, these levels were higher than those elicited by stimulation with EB and therefore, cannot be used as a parameter indicative of protection.We conclude that the cellular immune responses were similar in mice immunized with CAF01 or CAF09.Also, no significant differences were observed between groups vaccinated with nMOMP versus rMOMP.

Changes in body weight of mice following the i.n. challenge
As a measurement of the systemic effect of the infection, the body weight was determined for 10 days following the i.n.challenge.All mice, except those immunized i.n. with EB, lost weight for the first 2-4 days post challenge (d.p.c.) (Fig. 2).Subsequently, OD at 405nm mice vaccinated with CAF01, or CAF09 and nMOMP, or rMOMP slowly regained most of their initial body weight in contrast to controls immunized with MEM (P < 0.05).As determined by the repeated measures ANOVA test, the cumulative body weight changes over the 10-day period were significantly (P < 0.05) different between mice immunized with CAF01 plus nMOMP and the other three experimental groups.No significant differences were observed between the CAF09 nMOMP versus rMOMP group (P > 0.05).By 10 d.p.c., mice immunized with CAF01 and nMOMP, or rMOMP, weighted À4.02 ± 0.59% and À6.32 ± 1.11% less, respectively, than their initial body weight (Fig. 3A and Table 3).Similarly, mice immunized with CAF09 and nMOMP, or rMOMP, weighted À5.63 ± 1.06% and À4.46 ± 1.81% less, respectively than their initial body weight.None of these body weight changes are significantly different from each other (P > 0.05).These body weight changes at 10 d.p.c., however, are significantly different when compared mice inoculated with MEM (À19.27 ± 1.86%) (P < 0.05).Therefore, both CAF01 and CAF09, formulated with nMOMP or rMOMP, were similarly efficient at protecting mice from the systemic effects of C. muridarum infection.

Lungs weight
As a parameter of local inflammatory responses, the lungs weight was determined at 10 d.p.c.(Fig. 3B and Table 3) The mean weight of the lungs (g) in the two groups of mice vaccinated with CAF01 (0.23 ± 0.01), or CAF09 (0.25 ± 0.01) and nMOMP was significantly lower than MEM immunized controls (0.30 ± 0.01; P < 0.05) but not significantly different among themselves (P > 0.05).In animals vaccinated with CAF01 (0.33 ± 0.01), or CAF09 (0.32 ± 0.02), and rMOMP lungs weights were not significantly different among them (P > 0.05) and were similar to the MEM group.These results indicate that both CAF01 and CAF09 adjuvants were equally effective at inducing immune responses that helped cleared the local infection in the lungs.nMOMP was more effective than rMOMP at controlling local inflammatory responses.

Burden of C. muridarum infection in the lungs
Ten days after the i.n.challenge, mice were euthanized and their lungs cultured for C. muridarum (Fig 3C and Table 3).The median number of IFU recovered from lungs of mice vaccinated Cm EB = C. muridarum EB were incubated with irradiated splenocytes at a ratio of 5:1 for preparing antigen presenting cells.
Con A = Concanavalin A was used as positive stimulant at a concentration of 5 lg/ml.with CAF01 and nMOMP was 0.0002 (range: Â 10 6 and from those immunized with CAF09 was BLD (range: BLD-0.0005)Â 10 6 .These two groups were not significantly different among themselves.In comparison with the positive control inoculated with live EB, BLD (range BLD-BLD), the CAF09 group was not significantly different (P > 0.05) while the CAF01 group was (P < 0.05).The median number of IFU recovered from mice immunized with CAF01 and rMOMP was 7.1 (range: 0.007-82.8)Â 10 6 and those immunized with CAF09 3.6 (range: 0.037-61.9)Â 10 6 .The two groups immunized with rMOMP were not significantly different among themselves (P > 0.05) but were significantly different when compared with controls inoculated with EB (P < 0.05).
The number of IFU in the lungs of mice vaccinated with CAF01, or CAF09 using nMOMP or rMOMP, was significant lower than in mice immunized with MEM (median: 2463; range: 165-28,317) Â 10 6 IFU (P < 0.05).Therefore, protection against the local infection was similar for CAF01 and CAF09.nMOMP was more effective than rMOMP at clearing the lung infection.

Local immune responses in the lungs at 10 d.p.c
To evaluate local immune responses, the lung homogenates supernatants were collected at 10 d.p.c. and levels of IFN-c, IL-4 and C. muridarum-specific IgA were determined (Table The mean levels of IFN-c (pg/ml) in mice vaccinated with CAF01, or CAF09 and nMOMP were the same as those immunized with live EB (<15) indicative that, by 10 d.p.c., these animals had controlled the C. muridarum infection.These values are significantly different from those of mice immunized CAF01 (871 ± 216), or CAF09 (965 ± 315) and rMOMP, or MEM (2199 ± 238), that still had high amounts of C. muridarum IFU in their lungs (P < 0.05).No specific trends were observed in levels of IL-4 suggesting that this cytokine does not play a critical role in chlamydial infection or, that by 10 d. p.c., it has almost reached basal levels in all four groups.
The amounts of C. muridarum-specific IgA (OD 405 ) followed the opposite trend than the levels of IFN-c.Mice vaccinated with CAF01, or CAF09, all had higher levels of IgA than controls inoculated with MEM.Well-protected animals, in particular those vaccinated with CAF01 (2.69 ± 0.11), or CAF09 (2.32 ± 0.15) and nMOMP and the controls immunized with EB (3.05 ± 0.04) had very high levels of IgA.
In summary, both CAF01 and CAF09 were similarly effective at controlling the local infection in the lungs.nMOMP was more efficacious than rMOMP at eliciting protective immune responses.

Discussion
The adjuvants CAF01 and CAF09, mixed with C. muridarum nMOMP or rMOMP, were compared for their ability to elicit protective immune responses in BALB/c mice against an i.n.challenge.Qualitative and quantitative similar humoral and cellular immune responses were elicited by both adjuvants.As determined by changes in body weight, lungs weight and number of C. muridarum IFU recovered from lungs, both adjuvants induced comparable protection.With both adjuvants, more robust protection was elicited with nMOMP than with rMOMP.
Here, CAF01 and CAF09 were formulated with the C. muridarum nMOMP, or rMOMP and BALB/c mice were immunized by the s.c.followed by the i.n.routes and challenged i.n. with 10 4 IFU of C. muridarum.Significant C. muridarum-specific total IgG antibody titers were detected in serum collected the day before the challenge from the four groups of mice.IgG antibody titers were higher in mice immunized with nMOMP than rMOMP.Both CAF01 and CAF09 favor Th1 responses [14,19,22].To determine the vaccinations elicited Th1, or Th2-biased responses, the IgG2a/IgG1 ratios were determined.Interestingly, both adjuvants favored Th1 immune responses when formulated with nMOMP (IgG2/IgG1 ratios: $2-4) while, with rMOMP, responses were more balanced (IgG2/IgG1 ratios: $0.3-1).Massari et al. [16] have shown that nMOMP proteosomes signal via TLR-2.Adjuvants that signal via TLR2 induce mostly Th2-type immune responses [17].Due to the structural heterogeneity, proteosomes cannot be produced with rMOMP and therefore, cannot be tested for adjuvant activity.Thus, we need to assume that rMOMP also has adjuvant activity and induces more robust Th2 responses than nMOMP.CAF01 and CAF09 formulated with nMOMP, but not with rMOMP, elicited neutralizing antibodies that likely accounted for better protection.
Using synthetic peptides we determined MOMP domains recognized by IgG antibodies.As expected, most antibodies bound to the four VDs, although CD5 was also recognized [32].No major differences were observed between mice immunized with CAF01 versus CAF09 and rMOMP although mice receiving CAF01 only recognized epitopes in VD1 and CD5 while those immunized using CAF09 also recognized VD3 epitopes.Mice vaccinated using nMOMP, with both adjuvants, mounted broader antibody responses that included all the VDs, than those immunized with rMOMP.This may account for the better protection obtained with nMOMP since neutralizing B-cell epitopes are mainly located in VDs while T-cell epitopes map mainly to CDs [32][33][34].
Based on a T-cell lymphoproliferative assay and production of IFN-c, all four-vaccine formulations elicited robust Th-1 cell mediated immune responses.Interestingly, these responses were equivalent to those induced in positive control mice immunized with live EB.These results are very encouraging since in general, using other adjuvants, we have found the cellular immune responses to be weaker for vaccines formulated with MOMP, in particular rMOMP, than those of animals immunized with live EB [35].These results therefore, confirm the ability of both CAF01 and CAF09 to elicit strong Th1-biased immune responses in mice using antigens from various pathogens including malaria, Mycobacterium tuberculosis and C. muridarum [20,23,[36][37][38].
As shown by body weight loss, the four groups of mice, immunized with CAF01 or CAF09 and MOMP, were protected against the i.n. C. muridarum challenge when compared with the MEMimmunized group.Weight loss occurred mainly on days 2 to 3 p.c.During this time period, the C. muridarum infectious challenge likely goes through the first round of replication and systemic dissemination.The body weight then slowly increased over the next 6 d. indicative of control of the infection.In contrast, negative controls vaccinated with MEM continuously lost weight during the 10 d. of observation.As expected, positive controls immunized with EB had minimal body weight loss by 3 d.p.c. and then regained their initial weight.
Lungs weight showed that only mice vaccinated with CAF01, or CAF09, and nMOMP were protected.This suggests that while mice vaccinated with nMOMP had already controlled the local growth of C. muridarum by 10 d.p.c., those immunized with rMOMP still had significant inflammation.This was confirmed when the bacterial load was determined in lungs.As shown by the number of C. muridarum IFU recovered from the lungs, vaccination with CAF01 or CAF09 and nMOMP, elicited equivalent protection to EB. Groups that received rMOMP were also protected but had significantly higher numbers of IFU than those vaccinated with nMOMP.Levels of IFN-c in lungs supernatants, used as an indication of local inflammatory responses, paralleled these results.Groups of mice with high numbers of IFU in lungs had high levels of IFN-c while those with low IFU number had low IFN-c levels.Titers of C. muridarum specific IgA followed the opposite pattern suggesting that this local immune response has an important role in protection.The somewhat stronger IgA by the CAF01 formulation compared to CAF09, is likely relate to the ability of CAF01 to promote IL-17 responses that have been reported to accelerate the recruitment of IgA producing B cells and upregulate polymeric immunoglobulin receptor (pIgR) expression in the mucosal epithelium of importance for IgA secretion [24,25,39].
In summary, CAF01 and CAF09, formulated with MOMP, elicited similarly strong protective humoral and cell mediated immune responses in mice against a C. muridarum i.n.challenge.nMOMP induced a more robust protection than rMOMP.Since the cellular immune responses were equivalent in mice vaccinated with nMOMP versus rMOMP while antibody levels were overall higher, qualitatively broader and had more neutralizing activity in animals immunized with nMOMP than rMOMP, we conclude that antibody responses may account for some of the differences in protection between the two MOMP preparations.These findings support experiments, in which a critical role for antibodies in protection against a chlamydial challenge was determined [14,18,19,40].The more robust protection induced by nMOMP versus rMOMP, expands studies in which other adjuvants were used, and confirms the critical role in protection played by the structural conformation of MOMP [35,41].

Vaccine 35 (
2017) 1705-1711 Contents lists available at ScienceDirect Vaccine j o u r n a l h o m e p a g e : w w w .e l s e v i e r .c o m / l o c a t e / v a c c i n e Overlapping 25 aa peptides of C. muridarum MOMP VD1 VD2 VD3 VD4

Fig. 1 .
Fig. 1.Binding of serum antibodies to synthetic C. muridarum MOMP peptides.Serum samples from immunized mice were collected the day before the i.n.challenge and their reactivity to 25-mer peptides corresponding to the C. muridarum mature MOMP were analyzed by ELISA.

Fig. 3 .
Fig. 3. (A) Percentage change in mean body weight at 10 d. following the i.n.challenge with C. muridarum.The mean is shown as a horizontal line.Each symbol represents a single animal.* : P < 0.05 when compared to MEM-immunized group.(B) Lungs weight at 10 d. following the i.n.challenge with C. muridarum.The mean is shown as a horizontal line.Each symbol represents a single animal.* : P < 0.05 when compared to other-immunized group.(C) Number of IFU recovered from the lungs at 10 d. following the i.n.challenge with C. muridarum.The median is shown as a horizontal line.Each symbol represents a single animal.* : P < 0.05 when compared to other-immunized group.

Table 1
Humoral immune responses in sera and vaginal washes the day before challenge.

Table 2
In vitro T cell proliferative response and cytokine production from stimulated T cells the day before challenge.