Potential of DPD ((S)-4,5-dihydroxy-2,3-pentanedione) Analogs in Microparticulate Formulation as Vaccine Adjuvants

The molecule (S)-4,5-dihydroxy-2,3-pentanedione (DPD) is produced by many different species of bacteria and is involved in bacterial communication. DPD is the precursor of signal molecule autoinducer-2 (AI-2) and has high potential to be used as a vaccine adjuvant. Vaccine adjuvants are compounds that enhance the stability and immunogenicity of vaccine antigens, modulate efficacy, and increase the immune response to a particular antigen. Previously, the microparticulate form of (S)-DPD was found to have an adjuvant effect with the gonorrhea vaccine. In this study, we evaluated the immunogenicity and adjuvanticity of several synthetic analogs of the (S)-DPD molecule, including ent—DPD((R)-4,5-dihydroxy-2,3-pentanedione), n-butyl—DPD ((S)-1,2-dihydroxy-3,4-octanedione), isobutyl—DPD ((S)-1,2-dihydroxy-6-methyl-3,4-heptanedione), n-hexyl—DPD ((S)-1,2-dihydroxy-3,4-decanedione), and phenyl—DPD ((S)-3,4-dihydroxy-1-phenyl-1,2-butanedione), in microparticulate formulations. The microparticulate formulations of all analogs of (S)-DPD were found to be noncytotoxic toward dendritic cells. Among these analogs, ent—DPD, n-butyl—DPD, and isobutyl—DPD were found to be immunogenic toward antigens and showed adjuvant efficacy with microparticulate gonorrhea vaccines. It was observed that n-hexyl—DPD and phenyl—DPD did not show any adjuvant effect. This study shows that synthetic analogs of (S)-DPD molecules are capable of eliciting adjuvant effects with vaccines. A future in vivo evaluation will further confirm that these analogs are promising vaccine adjuvants.


Introduction
Vaccination is used to generate a strong immune response against a particular pathogen, and this response may provide long-term protection against an infection.Adjuvants are compounds added to vaccine formulations to enhance the response against co-inoculated antigens.The word adjuvant originates from the Latin word adjuvare, which means 'to help or to enhance' [1][2][3].Functionally, adjuvants increase the immunogenicity of antigens, speed up the mechanism, enhance the duration of the immune response, stimulate cell-mediated immunity, promote the induction of mucosal immunity, amplify the immune response in immunologically immature individuals, and reduce the dose of antigen necessary, lowering vaccine costs [4,5].Traditional vaccines use weakened or inactivated pathogens as antigens that are heterogeneous and contain many epitopes.Some of these epitopes provide an extended T-cell response, but their contribution to immunogenicity is minuscule (6).For instance, mRNA vaccines offer more benefits than traditional vaccines such as inexpensive production, easy scale-up, extended safety, and high-level antigen expression.There are still several unanswered questions about mRNA vaccines including challenges associated with the vaccines' development and use [6].There are several types of current vaccines, and their advantages and limitations are shown in Table 1 [7,8].Therefore, the other hand, at a high cell density (HCD), the production of AIs increases and results in a high concentration that causes the initiation of detection and response.Secondly, at high concentrations, the AIs are detected by receptors that exist in the cytoplasm or in the membrane.Finally, the detection of AI by neighboring bacteria becomes activated and produces its own AI to further increase the communication process [21].
In previous studies conducted in our lab, we evaluated the potential of the (S)-DPD molecule in microparticulate form as a possible vaccine adjuvant.We found that microparticulate (S)-DPD acted as an adjuvant with a microparticulate gonorrhea vaccine [23].Therefore, in this study, as a continuation of our previous finding, we investigated the potential of synthetic analogs of (S)-DPD as probable vaccine adjuvants.The synthetic analog molecules of (S)-DPD that have been tested are described in Figure 1.
density (LCD) of the bacterial population, AIs diffuse away, which is present at lower concentrations.On the other hand, at a high cell density (HCD), the production of AIs increases and results in a high concentration that causes the initiation of detection and response.Secondly, at high concentrations, the AIs are detected by receptors that exist in the cytoplasm or in the membrane.Finally, the detection of AI by neighboring bacteria becomes activated and produces its own AI to further increase the communication process [21].
In previous studies conducted in our lab, we evaluated the potential of the (S)-DPD molecule in microparticulate form as a possible vaccine adjuvant.We found that microparticulate (S)-DPD acted as an adjuvant with a microparticulate gonorrhea vaccine [23].Therefore, in this study, as a continuation of our previous finding, we investigated the potential of synthetic analogs of (S)-DPD as probable vaccine adjuvants.The synthetic analog molecules of (S)-DPD that have been tested are described in Figure 1.The development of an effective gonorrhea vaccine has been demanding because of the antigenic variability of N. gonorrhea and its ability to inhibit the development of adaptive immune responses.Previous endeavors to formulate an effective gonorrhea vaccine, either whole-cell a enuated or subunit recombinant, were unsuccessful.The clinical representation in symptomatic males versus female patients is different.In males, symptoms include urethral discharge, dysuria, and testicular or different levels of rectal pain.In females, symptoms include vaginal discharge, dysuria, dyspareunia, abnormal uterine bleeding, and lower abdominal and/or rectal pain.In terms of the pathological aspect, N. gonorrhoeae has the ability to colonize the genital mucosa.However, this infection can also colonize other mucosa such as ocular, nasopharyngeal, and anal mucosa.Once the immune system is activated, there can be further complications from untreated, ascending, genital-tract infections.In women, this infection can lead to pelvic inflammatory disease, infertility, and ectopic pregnancy [24,25].In this study, we used a gonorrhea vaccine that was previously formulated in our laboratory, in which whole-cell inactivated N. gonorrhea was entrapped in a polymer matrix of a microparticle formulation [26,27].The N. gonorrhea microparticle (MP) is shown in Figure 2. The development of an effective gonorrhea vaccine has been demanding because of the antigenic variability of N. gonorrhea and its ability to inhibit the development of adaptive immune responses.Previous endeavors to formulate an effective gonorrhea vaccine, either whole-cell attenuated or subunit recombinant, were unsuccessful.The clinical representation in symptomatic males versus female patients is different.In males, symptoms include urethral discharge, dysuria, and testicular or different levels of rectal pain.In females, symptoms include vaginal discharge, dysuria, dyspareunia, abnormal uterine bleeding, and lower abdominal and/or rectal pain.In terms of the pathological aspect, N. gonorrhoeae has the ability to colonize the genital mucosa.However, this infection can also colonize other mucosa such as ocular, nasopharyngeal, and anal mucosa.Once the immune system is activated, there can be further complications from untreated, ascending, genital-tract infections.In women, this infection can lead to pelvic inflammatory disease, infertility, and ectopic pregnancy [24,25].In this study, we used a gonorrhea vaccine that was previously formulated in our laboratory, in which whole-cell inactivated N. gonorrhea was entrapped in a polymer matrix of a microparticle formulation [26,27].The N. gonorrhea microparticle (MP) is shown in Figure 2. Depending on their function, adjuvants can be classified juvants or vaccine delivery systems [3,28,29].Immunostimula eral salts, immunostimulatory complexes, polysaccharides, a ucts [30].Vaccine delivery systems include liposomes, and m There are currently six types of FDA-approved adjuvants on monophosphoryl lipid A, oil-in-water emulsion, CpG, and Q num adjuvants are the most common adjuvants among the num adjuvants elicit several adverse effects that are related t Such effects include aluminum-induced injection site pain and reflect cell necrosis and the induction of inflammasome act [35].
Particulate adjuvants have particle sizes comparable to ally, smaller particles (<5 µm) are more immunogenic than adjuvants can also modulate the type of immune response to gen and aid in cross-presentation of the antigen, which is a pr T-cell responses.Moreover, adding up such potent immun delivery systems may reduce adverse events by limiting the adjuvant [36][37][38].By formulating the various analogs of (S)-D are consolidating the advantages of both classes of adjuvants The microparticles were formulated using the biocompa Depending on their function, adjuvants can be classified as immunostimulatory adjuvants or vaccine delivery systems [3,28,29].Immunostimulatory adjuvants include mineral salts, immunostimulatory complexes, polysaccharides, and microbial-derived products [30].Vaccine delivery systems include liposomes, and micro-and nanoparticles [31].There are currently six types of FDA-approved adjuvants on the market: aluminum salts, monophosphoryl lipid A, oil-in-water emulsion, CpG, and QS-21 saponin [32,33].Aluminum adjuvants are the most common adjuvants among the marketed products.Aluminum adjuvants elicit several adverse effects that are related to their mechanism of action.Such effects include aluminum-induced injection site pain and tenderness [34], which may reflect cell necrosis and the induction of inflammasome activation and IL-1 production [35].
Particulate adjuvants have particle sizes comparable to those of pathogens.Generally, smaller particles (<5 µm) are more immunogenic than larger particles.Particulate adjuvants can also modulate the type of immune response toward the co-inoculated antigen and aid in cross-presentation of the antigen, which is a prime role for generating CD8 + T-cell responses.Moreover, adding up such potent immunostimulatory adjuvants into delivery systems may reduce adverse events by limiting the systemic circulation of the adjuvant [36][37][38].By formulating the various analogs of (S)-DPD into microparticles, we are consolidating the advantages of both classes of adjuvants.
The microparticles were formulated using the biocompatible and biodegradable polymer PLGS (poly (lactic-co-glycolic acid)) matrix.PLGA particles are known to increase the potency of vaccine and adjuvant formulations.Previously, several investigations have indicated the significance of PLGA particulate vaccines in stimulating robust Th1-type immune responses that are detected by the secretion of IgG2a antibodies.Moreover, these adjuvant-containing particles enhance and prolong the cross-presentation efficiency [39][40][41].
In this study, we evaluated the adjuvant effect of microparticulate formulations loaded with various (S)-DPD analogs.We initially tested these microparticulate formulations of analogs for their cytotoxicity and immunogenicity profiles, which was followed by evaluating their adjuvant potential upon combination with the microparticulate gonorrhea vaccine that was previously developed in our laboratory.We compared the adjuvant potential of these microparticulate formulations with the adjuvant effect of FDA-approved adjuvants as microparticulate formulations.

Microparticle Formulation and Quatitation
The percentage of yield of the microparticulate formulations of various DPD analogs ranged from 86 to 92% (Table 2).The loss of particles can be attributed to material loss during homogenization and centrifugation.The numbers in Table 2 represent the parameters of percentage of yield (%), size of particles (nm), polydispersity index (PDI), and surface zeta potential (mV) of particles.The recovery yield is measured to evaluate the percent of product.In this study, all the DPD analogs showed a higher recovery yield.The particle size is important for antigen uptake by antigen-presenting cells (APCs).The polydispersity index (PDI) is an indicator of the uniform size distribution of the particles.In Table 1, for all analogs, the results showed a lower PDI which indicates that our microparticles were of uniform size distribution.We also measured the zeta or charge of the MPs.The charges for all the DPD analogs were negative.The negative surface charge indicates the elevated stability of microparticles in aqueous solution.

Particle Size Analysis
A Malvern Nano Zetasizer (ZS) was used to measure the particle size of various microparticulate formulations loaded with different analogs of (S)-DPD.The particle size ranged from 350 to 500 nm (Table 1).We observed no significant differences between the size of microparticulate formulations and blank (unloaded) microparticles (n = 6).

Zeta Potential Measurement
The surface zeta potential value of the DPD analog-loaded microparticles was determined by using a Malvern Nano ZS and ranged from −30 to −35 mV (Table 1).No significant difference between the zeta potential of the blank (unloaded) microparticles and DPD analog-loaded microparticles was observed.

Scanning Electron Microscopy (SEM) Analysis
In scanning electron microscopy (SEM), the surface morphology of the DPD analogloaded microparticles was pictured (Figure 3).The shape of the microparticles was found to be spherical with no deformities.The SEM figures show that the particles were aggregated.We assume that the colloidal stability of the biodegradable PLGA strongly affected in aqueous solution which may have resulted in the aggregation of particles [42].

Griess's Assay Analysis
Nitric oxide release is an important marker used to assess the induction of the innate immune response.Increased nitric oxide release indicates an increased immune response induction.In this study, nitric oxide release from exposed dendritic cells was set up as a positive control group, and only dendritic cells were used as the negative control group.The nitric oxide release from blank microspheres, the DPD solution, and DPD and its analogs with adjuvant alum and MF59 MP was also determined.The nitric oxide release from dendritic cells exposed to the positive control group (marketed measles vaccine) was comparable to that from the dendritic cells exposed to microparticulate ent-DPD, n-butyl-DPD, and isobutyl-DPD.However, the cells exposed to n-hexyl-DPD and phenyl-DPD microparticles released significantly lower amounts of nitric oxide when compared to those exposed to the positive control (Figure 4).This indicates that the microparticulate formulations of ent-DPD, n-butyl-DPD, and isobutyl-DPD had an immunogenicity equivalent to that of the marketed measles vaccine, while the immunogenicity of n-hexyl-DPD and phenyl-DPD microparticles was lower.

Griess's Assay Analysis
Nitric oxide release is an important marker used to assess the induction of the innate immune response.Increased nitric oxide release indicates an increased immune response induction.In this study, nitric oxide release from exposed dendritic cells was set up as a positive control group, and only dendritic cells were used as the negative control group.The nitric oxide release from blank microspheres, the DPD solution, and DPD and its analogs with adjuvant alum and MF59 MP was also determined.The nitric oxide release from dendritic cells exposed to the positive control group (marketed measles vaccine) was comparable to that from the dendritic cells exposed to microparticulate ent-DPD, n-butyl-DPD, and isobutyl-DPD.However, the cells exposed to n-hexyl-DPD and phenyl-DPD microparticles released significantly lower amounts of nitric oxide when compared to those exposed to the positive control (Figure 4).This indicates that the microparticulate formulations of ent-DPD, nbutyl-DPD, and isobutyl-DPD had an immunogenicity equivalent to that of the marketed measles vaccine, while the immunogenicity of n-hexyl-DPD and phenyl-DPD microparticles was lower.

Cytotoxicity Study
The microparticulate DPD analogs (ent-DPD, n-butyl-DPD, isobutyl-DPD, n hexyl-DPD, and phenyl-DPD) were noncytotoxic toward dendritic cells at all teste concentrations (50 µg/mL to 500 µg/mL) (Figure 5).There was no significant difference between nitric oxide released from marketed measles vaccine compared to ent-DPDMPs, n-butyl-DPD MPs, and isobutyl-DPD MPs.However, cells exposed to marketed measles vaccine released remarkably higher amounts of nitric oxide than the groups of cells exposed to n-hexyl-DPD MPs and phenyl-DPD MPs.Results are presented as the mean ± SEM (n = 3).*** p < 0.001 extremely significant.

Cytotoxicity Study
The microparticulate DPD analogs (ent-DPD, n-butyl-DPD, isobutyl-DPD, nhexyl-DPD, and phenyl-DPD) were noncytotoxic toward dendritic cells at all tested concentrations (50 µg/mL to 500 µg/mL) (Figure 5).Nitrite level in supernatant was measured using Griess's reagents.There was no significant difference between nitric oxide released from marketed measles vaccine compared to ent-DPDMPs, n-butyl-DPD MPs, and isobutyl-DPD MPs.However, cells exposed to marketed measles vaccine released remarkably higher amounts of nitric oxide than the groups of cells exposed to n-hexyl-DPD MPs and phenyl-DPD MPs.Results are presented as the mean ± SEM (n = 3).*** p < 0.001 extremely significant.

Expression of Antigen-Presenting Molecules
In the in vitro study, it was detected that the dendritic cells exposed to ent-DPD, nbutyl-DPD, and isobutyl-DPD microparticles showed a comparable expression of the MHC I and MHC II antigen-presenting molecules as the cells exposed to the marketed measles vaccine (positive control).However, the dendritic cells showed significantly lower expression upon exposure to the n-hexyl-DPD microparticles and phenyl-DPD microparticles compared to the positive control.This showed that the immunogenicity of ent-DPD, n-butyl-DPD, and isobutyl-DPD microparticles was comparable to that of the marketed measles vaccine, while n-hexyl-DPD microparticles and phenyl-DPD microparticles were significantly less immunogenic than the vaccine (Figure 7).A 50% release of ent-DPD was observed from the PLGA polymer at 48 h.All other analogs displayed a slower release profile than ent-DPD (50% release at approximately 168 h).

Expression of Antigen-Presenting Molecules
In the in vitro study, it was detected that the dendritic cells exposed to ent-DPD, n-butyl-DPD, and isobutyl-DPD microparticles showed a comparable expression of the MHC I and MHC II antigen-presenting molecules as the cells exposed to the marketed measles vaccine (positive control).However, the dendritic cells showed significantly lower expression upon exposure to the n-hexyl-DPD microparticles and phenyl-DPD microparticles compared to the positive control.This showed that the immunogenicity of ent-DPD, n-butyl-DPD, and isobutyl-DPD microparticles was comparable to that of the marketed measles vaccine, while n-hexyl-DPD microparticles and phenyl-DPD microparticles were significantly less immunogenic than the vaccine (Figure 7).

Expression of Costimulatory Molecules
There was no significant difference in terms of the expression of the costimulatory molecule CD40 between the cells that were exposed to the marketed measles vaccine and ent-DPD and n-butyl-DPD microparticles.However, the cells exposed to isobutyl-DPD, n-hexyl-DPD, and phenyl-DPD microparticles showed a significantly lower expression of CD40 than the cells exposed to the marketed vaccines.This indicates that the ent-DPD and n-butyl-DPD microparticles had an immunogenicity comparable to that of the mar-keted measles vaccine.However, there was a significant difference in the immunogenicity between the marketed measles vaccine and analogs of isobutyl-DPD, n-hexyl-DPD, and phenyl-DPD microparticles (Figure 8A).

Expression of Costimulatory Molecules
There was no significant difference in terms of the expression of the costimulatory molecule CD40 between the cells that were exposed to the marketed measles vaccine and ent-DPD and n-butyl-DPD microparticles.However, the cells exposed to isobutyl-DPD, n-hexyl-DPD, and phenyl-DPD microparticles showed a significantly lower expression of CD40 than the cells exposed to the marketed vaccines.This indicates that the ent-DPD and n-butyl-DPD microparticles had an immunogenicity comparable to that of the marketed measles vaccine.However, there was a significant difference in the immunogenicity between the marketed measles vaccine and analogs of isobutyl-DPD, nhexyl-DPD, and phenyl-DPD microparticles (Figure 8A).

Expression of Costimulatory Molecules
There was no significant difference in terms of the expression of the costimulatory molecule CD40 between the cells that were exposed to the marketed measles vaccine and ent-DPD and n-butyl-DPD microparticles.However, the cells exposed to isobutyl-DPD, n-hexyl-DPD, and phenyl-DPD microparticles showed a significantly lower expression of CD40 than the cells exposed to the marketed vaccines.This indicates that the ent-DPD and n-butyl-DPD microparticles had an immunogenicity comparable to that of the marketed measles vaccine.However, there was a significant difference in the immunogenicity between the marketed measles vaccine and analogs of isobutyl-DPD, nhexyl-DPD, and phenyl-DPD microparticles (Figure 8A).).Dendritic cells showed significantly lower expression upon exposure to the n-hexyl-DPD microparticles and phenyl-DPD microparticles compared to the positive control.The term "There was no significant difference between" was used to compare the different groups based on the results of the statistical test.In Figure Figure 8. Costimulatory molecules CD40 and CD80.Level of (A) CD40 and (B) CD80 on the surface of murine dendritic cells.Results are expressed as mean ± SEM (n = 3).Dendritic cells showed significantly lower expression upon exposure to the n-hexyl-DPD microparticles and phenyl-DPD microparticles compared to the positive control.The term "There was no significant difference between" was used to compare the different groups based on the results of the statistical test.In Figure 7, for instance, there was no significant difference between the marketed measles vaccine versus the ent-DPDMPs and n-butyl-DPD MPs.This indicates that even though non-significant, ent-DPDMPs and n-butyl-DPD MPs showed similar immunostimulatory potential as the positive control, which was a marketed vaccine.In this manner, the DPD analogs ent-DPD and n-butyl-DPD worked similarly to the marketed vaccine.* p < 0.05, ** p < 0.01, *** p < 0.001 extremely significant.
For the expression of the costimulatory molecule CD80, there was no significant difference between the cells exposed to the marketed measles vaccine and those exposed to the ent-DPD microparticles.However, the cells exposed to n-butyl-DPD, isobutyl-DPD, n-hexyl-DPD, and phenyl-DPD microparticles showed significantly lower expression of the costimulatory molecule CD80 than the cells exposed to the marketed measles vaccine.This indicates that the DPD microparticles have an immunogenicity comparable to that of the marketed measles vaccine.However, the immunogenicity of n-butyl-DPD, isobutyl-DPD, n-hexyl-DPD, and phenyl-DPD microparticles was significantly lower than that of the marketed measles vaccine (Figure 8B).

Evaluation of Adjuvant Effect: Griess's Assay for Nitrite
Nitric oxide release from antigen-presenting cells (APCs) is a hallmark of the induction of the innate immune response (32).In this study, it was found that when the dendritic cells were faced with gonorrhea vaccine microparticles combined with ent-DPD, n-butyl-DPD, and isobutyl-DPD microparticles, they released remarkably higher amounts of nitric oxide than the cells faced with gonorrhea vaccine microparticles only.However, there were no notable differences observed in terms of nitric oxide released from the cells faced with the combination of gonorrhea vaccine microparticles, n-hexyl-DPD microparticles, and phenyl-DPD microparticles in comparison with the cells that faced only gonorrhea vaccine microparticles.(Figure 9).Thus, ent-DPD, n-butyl-DPD, and isobutyl-DPD microparticles show an adjuvant effect with gonorrhea vaccine microparticles.
ference between the cells exposed to the marketed measles vaccine and tho the ent-DPD microparticles.However, the cells exposed to n-butyl-DP DPD, n-hexyl-DPD, and phenyl-DPD microparticles showed significan pression of the costimulatory molecule CD80 than the cells exposed to the m sles vaccine.This indicates that the DPD microparticles have an immunoge rable to that of the marketed measles vaccine.However, the immunogenicit DPD, isobutyl-DPD, n-hexyl-DPD, and phenyl-DPD microparticles wa lower than that of the marketed measles vaccine (Figure 8B).

Evaluation of Adjuvant Effect: Griess's Assay for Nitrite
Nitric oxide release from antigen-presenting cells (APCs) is a hallmark tion of the innate immune response (32).In this study, it was found that w dritic cells were faced with gonorrhea vaccine microparticles combined w n-butyl-DPD, and isobutyl-DPD microparticles, they released remar amounts of nitric oxide than the cells faced with gonorrhea vaccine micro However, there were no notable differences observed in terms of nitric o from the cells faced with the combination of gonorrhea vaccine micropartic DPD microparticles, and phenyl-DPD microparticles in comparison with faced only gonorrhea vaccine microparticles.(Figure 9).Thus, ent-DPD, n and isobutyl-DPD microparticles show an adjuvant effect with gonorrhe croparticles.

Evaluation of Adjuvant Effect with Bacterial and Viral Vaccines: Griess's Assay for Nitrite
We further tested the MP adjuvants with the analogs of ent-DPD, n-butyl-DPD, isobutyl-DPD, n-hexyl-DPD, and phenyl-DPD for their adjuvant potential by combining them with several bacterial and viral vaccines.We evaluated the adjuvant potential using two bacterial vaccines: MenAfriVac (MAV), a meningococcal A conjugate microparticle (Figure 10), and the Hemophilus influenzae type B (HIB) conjugate microparticle (Figure 11).We also evaluated the adjuvant potential with the following viral vaccines: measles vaccine microparticles (Figure 12), influenza (solution) vaccine (Figure 13), and inactivated Zika microparticles (Figure 14).Three adjuvants, ent-DPD, n-butyl-DPD, and isobutyl-DPD, showed adjuvant potential when combined with the MAV MP vaccine.When the compounds were tested with the HIB MP vaccine, measles MP vaccine, and Zika MP vaccine, only ent-DPD produced significant differences from the vaccine alone.When the MP adjuvants were combined with the influenza (S) vaccine, ent-DPD produced a significant response versus the vaccine alone.In addition, the influenza (S) vaccine and n-butyl MP produced similar responses.The results are shown in the following figures.combining them with several bacterial and viral vaccines.We evaluated the adjuvant potential using two bacterial vaccines: MenAfriVac (MAV), a meningococcal A conjugate microparticle (figure 10), and the Hemophilus influenzae type B (HIB) conjugate microparticle (figure 11).We also evaluated the adjuvant potential with the following viral vaccines: measles vaccine microparticles (figure 12), influenza (solution) vaccine (figure 13), and inactivated Zika microparticles (figure 14).Three adjuvants, ent-DPD, n-butyl-DPD, and isobutyl-DPD, showed adjuvant potential when combined with the MAV MP vaccine.When the compounds were tested with the HIB MP vaccine, measles MP vaccine, and Zika MP vaccine, only ent-DPD produced significant differences from the vaccine alone.When the MP adjuvants were combined with the influenza (S) vaccine, ent-DPD produced a significant response versus the vaccine alone.In addition, the influenza (S) vaccine and n-butyl MP produced similar responses.The results are shown in the following figures.Release of nitric oxide from dendritic cells as calculated from Griess's assay.Results are expressed as mean ± SEM (n = 3).* p < 0.05 significant.

Evaluation of Adjuvant Effect with Measles Vaccine Microparticles
The following figure shows the adjuvant effect of Measles Vaccine Microparticles combined with all five types of DPD Analogs.Release of nitric oxide from dendritic cells as calculated from Griess's assay.Results are expressed as mean ± SEM (n = 3).* p < 0.05 significant.

Evaluation of Adjuvant Effect with Measles Vaccine Microparticles
The following figure shows the adjuvant effect of Measles Vaccine Microparticles combined with all five types of DPD Analogs.Release of nitric oxide from dendritic cells as calculated from Griess's assay.Results are expressed as mean ± SEM (n = 3).* p < 0.05 significant.

Evaluation of Adjuvant Effect with Measles Vaccine Microparticles
The following figure shows the adjuvant effect of Measles Vaccine Microparticles combined with all five types of DPD Analogs.

Evaluation of Adjuvant Effect with Influenza (S) Vaccine
The following figure shows the adjuvant effect of Influenza Vaccine combined with all five types of DPD Analogs.

Evaluation of Adjuvant Effect: Expression of Antigen-Presenting Molecules
Dendritic cells exposed to the combination of the microparticulate gonorrhea vaccine and ent-DPD microparticles and n-butyl-DPD microparticles displayed a notably higher level

Evaluation of Adjuvant Effect with Inactivated Zika Vaccine Microparticles
The following figure shows the adjuvant effect of Zika Vaccine combined with all five types of DPD Analogs.
The following figure shows the adjuvant effect of Influenza Vaccine combined with all five types of DPD Analogs.

Evaluation of Adjuvant Effect: Expression of Antigen-Presenting Molecules
Dendritic cells exposed to the combination of the microparticulate gonorrhea vaccine and ent-DPD microparticles and n-butyl-DPD microparticles displayed a notably higher level

Evaluation of Adjuvant Effect: Expression of Antigen-Presenting Molecules
Dendritic cells exposed to the combination of the microparticulate gonorrhea vaccine and ent-DPD microparticles and n-butyl-DPD microparticles displayed a notably higher level of the antigen-presenting MHC I molecule than those exposed to only gonorrhea vaccine microparticles.However, there was no significant difference in MHC I molecule expression between the cells exposed to isobutyl-DPD, n-hexyl-DPD and phenyl-DPD microparticles combined with the microparticulate gonorrhea vaccine in contrast to the cells exposed to only microparticulate gonorrhea vaccine (Figure 15A).This fact reveals that the ent-DPD and n-butyl-DPD microparticles have an adjuvant effect with the microparticulate gonorrhea vaccine, while isobutyl-DPD, n-hexyl-DPD, and phenyl-DPD microparticles do not. of the antigen-presenting MHC I molecule than those exposed to only gonorrhea vaccine microparticles.However, there was no significant difference in MHC I molecule expression between the cells exposed to isobutyl-DPD, n-hexyl-DPD and phenyl-DPD microparticles combined with the microparticulate gonorrhea vaccine in contrast to the cells exposed to only microparticulate gonorrhea vaccine (Figure 15A).This fact reveals that the ent-DPD and nbutyl-DPD microparticles have an adjuvant effect with the microparticulate gonorrhea vaccine, while isobutyl-DPD, n-hexyl-DPD, and phenyl-DPD microparticles do not.We observed significantly higher expression of MHC II molecules from dendritic cells when exposed to the microparticulate formulations of ent-DPD, n-butyl-DPD, and isobutyl-DPD combined with the microparticulate gonorrhea vaccine in contrast to the cells exposed to only the microparticulate gonorrhea vaccine.However, no remarkable difference was observed in the expression of the antigen-presenting MHC II molecules between the cells exposed to the microparticulate formulations of n-hexyl-DPD and phenyl-DPD in combination with the gonorrhea vaccine microparticles compared to gonorrhea vaccine microparticles only (Figure 15B).Thus, microparticles of ent-DPD, n-butyl-DPD, and isobutyl-DPD were seen to show an adjuvant effect, while a similar adjuvant effect was not observed with n-hexyl-DPD and phenyl-DPD microparticles.

Evaluation of Adjuvant Effect: Expression of Costimulatory Molecules
The murine dendritic cells (DC 2.4) showed a significantly higher expression of CD40 upon exposure to the microparticulate formulations of the ent-DPD, n-butyl-DPD, and isobutyl-DPD microparticles integrated with the gonorrhea vaccine microparticles in contrast to the cells that were exposed to only gonorrhea vaccine microparticles.However, there was no significant difference in the expression of the costimulatory molecule CD40 on the surface of dendritic cells (DC 2.4) while exposed to only gonorrhea vaccine microparticles and combined with n-hexyl-DPD and phenyl-DPD microparticles (Figure 16A).Thus, microparticles of ent-DPD, n-butyl-DPD, and isobutyl-DPD showed an adjuvant effect, while a similar adjuvant effect was not observed with n-hexyl-DPD and phenyl-DPD microparticles.We observed significantly higher expression of MHC II molecules from dendritic cells when exposed to the microparticulate formulations of ent-DPD, n-butyl-DPD, and isobutyl-DPD combined with the microparticulate gonorrhea vaccine in contrast to the cells exposed to only the microparticulate gonorrhea vaccine.However, no remarkable difference was observed in the expression of the antigen-presenting MHC II molecules between the cells exposed to the microparticulate formulations of n-hexyl-DPD and phenyl-DPD in combination with the gonorrhea vaccine microparticles compared to gonorrhea vaccine microparticles only (Figure 15B).Thus, microparticles of ent-DPD, n-butyl-DPD, and isobutyl-DPD were seen to show an adjuvant effect, while a similar adjuvant effect was not observed with n-hexyl-DPD and phenyl-DPD microparticles.

Evaluation of Adjuvant Effect: Expression of Costimulatory Molecules
The murine dendritic cells (DC 2.4) showed a significantly higher expression of CD40 upon exposure to the microparticulate formulations of the ent-DPD, n-butyl-DPD, and isobutyl-DPD microparticles integrated with the gonorrhea vaccine microparticles in contrast to the cells that were exposed to only gonorrhea vaccine microparticles.However, there was no significant difference in the expression of the costimulatory molecule CD40 on the surface of dendritic cells (DC 2.4) while exposed to only gonorrhea vaccine microparticles and combined with n-hexyl-DPD and phenyl-DPD microparticles (Figure 16A).Thus, microparticles of ent-DPD, n-butyl-DPD, and isobutyl-DPD showed an adjuvant effect, while a similar adjuvant effect was not observed with n-hexyl-DPD and phenyl-DPD microparticles.The dendritic cells showed a significantly higher expression of the costimulatory molecule CD80 when exposed to the ent-DPD microparticles in combination with the gonorrhea vaccine microparticles.However, there was no significant difference in the expression of the costimulatory molecule CD80 on the surface of dendritic cells when exposed to the microparticulate formulations of n-butyl-DPD, isobutyl-DPD, n-hexyl-DPD, and phenyl-DPD in combination with the microparticulate gonorrhea vaccine compared to the cells exposed to the gonorrhea vaccine microparticles alone (Figure 16B).This shows that the ent-DPD microparticles have an adjuvant effect with gonorrhea vaccine microparticles, while the microparticulate formulations of n-butyl-DPD, isobutyl-DPD, n-hexyl-DPD, and phenyl-DPD do not.

Discussion
Bacteria use quorum sensing (QS), as a type of complex communication process, among themselves to control the population density.Through this process, bacteria act collectively as a group instead of as a single organism.The higher tolerance of bacteria to antibiotics is a ributed to the fact that bacteria can regulate its behavior through a QS communication process towards virulence and biofilm formation.Thus, disrupting this QS process could lead to the improved control of bacterial infections [19].Autoinducer-2, also known as a universal autoinducer, acts as a QS molecule that can act upon both types of bacteria, Gram-positive and Gram-negative.This molecule is derived from its precursor (S)-4,5-dihydroxy-2,3-pentanedione (DPD) molecule [20].
The AI-2 precursor (S)-DPD molecule can undergo a rapid interconversion to various linear and cyclic forms that are perceived by different types of bacteria, rendering QS targeting difficult.Thus, we previously tested the potential of a microparticulate formulation of the AI-2 precursor, (S)-DPD, as a potential vaccine adjuvant.Microparticulate (S)-DPD showed significant adjuvant activity with the microparticulate gonorrhea vaccine in in vitro studies [23].Based on these results, we further explored the potential of microparticulate formulations of various synthetic analogs of (S)-DPD (as described in Figure 1) as vaccine adjuvants with the microparticulate gonorrhea vaccine.
PLGA was chosen as the polymer matrix for the formulation of the microparticles.PLGA is a biocompatible and biodegradable material that provides effective protection to the encapsulated material [43,44].Equal amounts of blank microparticles were used as The dendritic cells showed a significantly higher expression of the costimulatory molecule CD80 when exposed to the ent-DPD microparticles in combination with the gonorrhea vaccine microparticles.However, there was no significant difference in the expression of the costimulatory molecule CD80 on the surface of dendritic cells when exposed to the microparticulate formulations of n-butyl-DPD, isobutyl-DPD, n-hexyl-DPD, and phenyl-DPD in combination with the microparticulate gonorrhea vaccine compared to the cells exposed to the gonorrhea vaccine microparticles alone (Figure 16B).This shows that the ent-DPD microparticles have an adjuvant effect with gonorrhea vaccine microparticles, while the microparticulate formulations of n-butyl-DPD, isobutyl-DPD, n-hexyl-DPD, and phenyl-DPD do not.

Discussion
Bacteria use quorum sensing (QS), as a type of complex communication process, among themselves to control the population density.Through this process, bacteria act collectively as a group instead of as a single organism.The higher tolerance of bacteria to antibiotics is attributed to the fact that bacteria can regulate its behavior through a QS communication process towards virulence and biofilm formation.Thus, disrupting this QS process could lead to the improved control of bacterial infections [19].Autoinducer-2, also known as a universal autoinducer, acts as a QS molecule that can act upon both types of bacteria, Gram-positive and Gram-negative.This molecule is derived from its precursor (S)-4,5-dihydroxy-2,3-pentanedione (DPD) molecule [20].
The AI-2 precursor (S)-DPD molecule can undergo a rapid interconversion to various linear and cyclic forms that are perceived by different types of bacteria, rendering QS targeting difficult.Thus, we previously tested the potential of a microparticulate formulation of the AI-2 precursor, (S)-DPD, as a potential vaccine adjuvant.Microparticulate (S)-DPD showed significant adjuvant activity with the microparticulate gonorrhea vaccine in in vitro studies [23].Based on these results, we further explored the potential of microparticulate formulations of various synthetic analogs of (S)-DPD (as described in Figure 1) as vaccine adjuvants with the microparticulate gonorrhea vaccine.
PLGA was chosen as the polymer matrix for the formulation of the microparticles.PLGA is a biocompatible and biodegradable material that provides effective protection to the encapsulated material [43,44].Equal amounts of blank microparticles were used as controls in all experiments.This ensured that the blank particles were nonimmunogenic, and, thus, the immunogenicity and adjuvanticity could be attributed to the analogs of (S)-DPD in the formulation [45].In the studies, the negative control was dendritic cells.However, the positive control, for instance, in Figure 3, was the marketed measles vaccine.Although there was a significant difference between the cells only group and the blank MP, the objective here was to compare the marketed vaccine versus our experiential adjuvant compounds.Generally, the blank MPs were expected to show a higher response than the cells only group, since the blank MP group was in a microparticulate form.This is one of the advantages of a microparticulate formulation.
As per the World Health Organization (WHO) guidelines, the probable adjuvant candidate should first be tested for its cytotoxic effect [46].Thus, we examined the cytotoxic effect of microparticulate formulations of various analogs of (S)-DPD toward dendritic cells using an MTT assay.We found that the microparticulate formulations of all the analogs of (S)-DPD tested were noncytotoxic toward dendritic cells.We performed a dose response for the microparticulate DPD analogs (ent-DPD, n-butyl-DPD, isobutyl-DPD, n-hexyl-DPD, and phenyl-DPD) in concentrations from 50 µg/mL (low) to 500 µg/mL (high).
In the cytotoxicity study, the objective was to determine an appropriate dose in vitro that could be used for further studies.In Figure 4, it shows that at low doses, such as 50 µg/mL, the DC or cell viability was very high.However, as the concentration increased, the cytotoxicity slowly increased.In the later studies, only a low dose was used considering the results found in the cytotoxicity study.In the past, from our laboratory, we have numerous published articles that showed an immunogenicity and cytotoxicity correlation between in vitro and in vivo assay, which indicated that the assays are biologically viable [38,39,43].
We further evaluated these microparticles for their ability to induce the innate immune response via a Griess's assay.In this study, the microparticulate formulations of ent-DPD, n-butyl-DPD, and isobutyl-DPD were identified to be immunogenic as they showed notable nitric oxide release levels from dendritic cells.In order to evaluate a formulation in vivo, it should be evaluated in vitro first.In an in vitro investigation, the immunostimulatory potential of these DPD analogs can be measured by measuring the NO release from dendritic cells.DC cells release NO and other cytokines after they are stimulated.NO is the first chemical that is released that we can measure in vitro that gives a great indication of what may happen in vivo.There are several studies that mention NO as an indicator for measuring an innate response.In the past from our laboratory, we have numerous published studies that showed an immunogenicity correlation between in vitro and in vivo assays [39,40,44].However, the microparticles of n-hexyl-DPD and phenyl-DPD were nonimmunogenic.The expression of antigen-presenting and costimulatory molecules also showed similar results.
The adjuvant potential evaluation of the MP adjuvants of analogs ent-DPD, n-butyl-DPD, isobutyl-DPD, n-hexyl-DPD, and phenyl-DPD for their adjuvant potential by combining them with several bacterial and viral vaccines also showed that some of the analogs have potential as adjuvants.MenAfriVac (MAV), a meningococcal A conjugate vaccine, was combined with several analogs of DPD (Figure 10).When tested with APCs, several adjuvants, including ent-DPD, n-butyl-DPD, and isobutyl-DPD, displayed similar immunostimulatory potential as the MAV.However, n-hexyl-DPD and phenyl-DPD did not produce significant NO release from APCs compared to the MAV vaccine alone or the positive control, LPS.The results indicated that the ent-DPD, n-butyl-DPD, and isobutyl-DPD MP adjuvants can be added to formulate an adjuvanted MAV vaccine.The second bacterial vaccine we evaluated was the Hemophilus influenzae type B (HIB) conjugate vaccine (Figure 11), which was combined with several adjuvants.Interestingly, only ent-DPD produced a significant response.However, the analog molecules n-butyl-DPD, isobutyl-DPD, n-hexyl-DPD, n-hexyl-DPD, and phenyl-DPD did not produce significant differences in comparison to the HIB vaccine alone.These results indicate that these small molecule microparticles may not produce a robust humoral or cellular immune response when formulated with the HIB vaccine.When several adjuvants were combined with the measles vaccine (Figure 12), only ent-DPD displayed a robust NO release, indicating that ent-DPD does have adjuvant potential.The other adjuvants, with analogs molecules n-butyl-DPD, isobutyl-DPD, n-hexyl-DPD, n-hexyl-DPD, and phenyl-DPD, did not produce a significant difference in NO compared to the measles vaccine alone.The results indicate that the measles vaccine can be combined with the ent-DPD adjuvant to formulate a vaccine.These compounds were then combined with the influenza (S) vaccine (Figure 13).Interestingly, only ent-DPD produced significantly higher NO compared to the vaccine alone.There was no adjuvant potential when the influenza (S) vaccine was combined with analog molecules of n-butyl-DPD, isobutyl-DPD, n-hexyl-DPD, and phenyl-DPD.Our results indicate that ent-DPD, combined with the influenza (S) vaccine, may produce a robust immune response.We also evaluated the immunostimulatory potential of our experimental microparticulate compounds with the inactivated Zika vaccine (Figure 14).For the Zika vaccine combined with several DPD analogs, only ent-DPD showed adjuvant potential in comparison to the vaccine alone.However, when the Zika vaccine was combined with analog molecules of n-butyl-DPD, isobutyl-DPD, n-hexyl-DPD, and phenyl-DPD, no significant benefit was observed, indicating that the addition of these two adjuvants may not further stimulate a robust immune response.In this case, a vaccine for the Zika virus can be formulated with adjuvant ent-DPD.Taken together, only ent-DPD was able to produce a significant NO level with several bacterial and viral vaccines.
Additionally, the induction of the innate immune response was evaluated as a measure of the adjuvant potential of the various microparticulate DPD analogs through exposure of the antigen-presenting cells to a combination of the microparticulate gonorrhea vaccine developed previously in our laboratory with various microparticulate DPD analogs [27].Microparticulate DPD analogs were weighed up to the microparticulate formulations of FDA-approved adjuvants, including alum and MF59 ® , in terms of their ability to act as an adjuvant.In this study, the microparticulate formulations of ent-DPD, n-butyl-DPD, and isobutyl-DPD were shown to display a remarkable adjuvant effect with the microparticulate gonorrhea vaccine.However, microparticulate formulations of n-hexyl-DPD and phenyl-DPD did not show an adjuvant effect.These results were found to be consistent with our results obtained for the immunogenicity assessment of the microparticulate formulations of (S)-DPD analogs.The expression of the antigen-presenting molecules MHC I and MHC II, as well as their corresponding costimulatory molecules CD80 and CD40, on the surface of dendritic cells upon exposure to the combination of microparticulate gonorrhea vaccine with various microparticulate DPD analogs further confirmed this effect.The microparticulate formulations of n-hexyl-DPD and phenyl-DPD did not show an adjuvant effect.However, the microparticles of ent DPD, n-butyl-DPD, and isobutyl-DPD did display an adjuvant effect, with ent-DPD microparticles showing the highest adjuvant effect with the microparticulate gonorrhea vaccine.
In our previous work, we assessed the bacterial QS biomolecule N-octanol-L-homoserine lactone, which is also known as autoinducer-1 (C8-HSL), as a potential vaccine adjuvant in vaccine formulations [43].In that study, the immunostimulatory potential of C8-HSL microparticles was compared with the current marketed FDA-approved microparticulate formulations of adjuvants such as Alhydrogel ® (alum), AddaVax™ (MF59), and cytosinephosphorothioate-guanine oligodeoxynucleotides (CpG).It was further determined that C8-HSL MP is immunostimulatory and produces an immune response similar to that of FDAapproved adjuvants (alum, MF59, and CpG).In addition, adjuvant efficacy was noted when the C8-HSL microparticle was integrated with several particulate vaccines, such as the Zika microparticle vaccine, measles microparticle vaccine, and marketed influenza vaccine [43].
The needs for future study include the investigation of adjuvant effects of microparticulate formulations of (S)-DPD analogs with an additional number of vaccines to confirm the adjuvanticity.Moreover, the concentration-dependent adjuvanticity of microparticulate formulations of (S)-DPD analogs and the in vivo study that will evaluate the other aspects of immunogenicity, such as antibody titers and cytokine level measurements that will provide clear insight into the safety, immunogenicity, and adjuvanticity of these formulations.At an early development stage, the microparticulate formulations of ent-DPD, n-butyl-DPD, and isobutyl-DPD were determined to be suitable adjuvant candidates regarding their formulation, non-cytotoxicity, and adjuvant effect.

Materials
The analogs of (S)-DPD were purchased from Omms Scientific Inc. (Dallas, TX, USA).Poly(vinyl alcohol) (PVA) and poly(lactic-co-glycolic acid) (PLGA) (50:50) were purchased from Sigma-Aldrich, St. Louis, MO, USA.Dendritic cells (DC 2.4) for in vitro study were obtained from ATCC.Adjuvants alum and AddaVax TM were purchased from InvivoGen, San Diego, CA, USA.The measles vaccine was obtained from the Serum Institute of India Pvt. Ltd.Pune, India.Antibodies used to stain murine MHC I, MHC II, CD40, and CD80 molecules for flow cytometric analysis were purchased from eBioscience laboratories (San Diego, CA, USA).Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS), penicillin/streptomycin, and nonessential amino acids were purchased from Cellgro Mediatech (Herndon, VA, USA).

Formulation of Microparticles
Microparticles were formulated using the double-emulsion solvent evaporation method [44,47,48].A 1% w/v solution of PLGA (50:50) was made by dissolving PLGA (50:50) in dichloromethane (DCM).Span 80 was added as an emulsifier.The aqueous solution of the various analogs of (S)-DPD (2% w/w) was then added and homogenized using the Omni TH Q probe homogenizer (18,000 rpm) equipment to make the primary W/O emulsion.The emulsion was then ensued by adding with 0.1% w/v solution of PVA and homogenized using a probe homogenizer (18,000 rpm) to form the W/O/W double emulsion.The formulation was kept in an ice bath throughout the process.The emulsion was kept on a magnetic stirrer for 5 h to completely evaporate the dichloromethane (DCM).The double emulsion was then ultracentrifuged to concentrate the microparticles.Trehalose was then added as the cryoprotectant, and the emulsion was lyophilized to obtain the dried microparticles (Figure 17).Furthermore, all three FDA approved adjuvants, Alum, MF59, and CpG, were made into microparticles.These microparticles were formulated using the double-emulsion solvent evaporation method as described here.
Pharmaceuticals 2023, 16, x FOR PEER REVIEW 20 of 25 microparticulate formulations of (S)-DPD analogs and the in vivo study that will evaluate the other aspects of immunogenicity, such as antibody titers and cytokine level measurements that will provide clear insight into the safety, immunogenicity, and adjuvanticity of these formulations.At an early development stage, the microparticulate formulations of ent-DPD, n-butyl-DPD, and isobutyl-DPD were determined to be suitable adjuvant candidates regarding their formulation, non-cytotoxicity, and adjuvant effect.

Formulation of Microparticles
Microparticles were formulated using the double-emulsion solvent evaporation method [44,47,48].A 1% w/v solution of PLGA (50:50) was made by dissolving PLGA (50:50) in dichloromethane (DCM).Span 80 was added as an emulsifier.The aqueous solution of the various analogs of (S)-DPD (2% w/w) was then added and homogenized using the Omni THQ probe homogenizer (18000 rpm) equipment to make the primary W/O emulsion.The emulsion was then ensued by adding with 0.1% w/v of PVA and homogenized using a probe homogenizer (18,000 rpm) to form the W/O/W double emulsion.The formulation was kept in an ice bath throughout the process.The emulsion was kept on a magnetic stirrer for 5 h to completely evaporate the dichloromethane (DCM).The double emulsion was then ultracentrifuged to concentrate the microparticles.Trehalose was then added as the cryoprotectant, and the emulsion was lyophilized to obtain the dried microparticles (Figure 17).Furthermore, all three FDA approved adjuvants, Alum, MF59, and CpG, were made into microparticles.These microparticles were formulated using the double-emulsion solvent evaporation method as described here.

Microparticle Recovery Yield
The recovery yield was determined for all the microparticles formulated.The following formula was employed to calculate the percent yield of microparticles:

Microparticle Recovery Yield
The recovery yield was determined for all the microparticles formulated.The following formula was employed to calculate the percent yield of microparticles:

Particle Size and Zeta Potential
To determine the size and zeta potential of the particles, 5 µg of microparticles was suspended in deionized water (1 mL).Then, the size and surface zeta potential of the microparticles were measured in a Malvern Nano ZS [19,31,38]

Scanning Electron Microscopy
The surface morphology of the microparticles loaded with various DPD analogs was visualized using scanning electron microscopy (SEM).Microparticles were dissolved in water and mounted using double-sided adhesive tape.Then, the surface morphology was evaluated using an SEM (scanning electron microscope) (Phenome benchtop SEM obtained from Nanoscience instruments, Phoenix, AZ, USA).

Griess's Assay for Nitrite
Nitric oxide release from the dendritic cells was measured to assess the immunogenicity of microparticles to trigger an immune response [24,49,50].Griess's assay was used to quantify nitrite, an oxidation product of nitric oxide, to evaluate the release of nitric oxide from dendritic cells.Various microparticulate formulations, including microparticles of ent DPD, n-butyl-DPD, isobutyl-DPD, n-hexyl-DPD, and phenyl-DPD, were incubated with murine dendritic cells (DC 2.4) (50 × 10 4 cells/well) in 48-well plates for 48 h.Blank (unloaded) microparticles and marketed measles vaccine were used as negative and positive controls, respectively.The solutions were then transferred to another plate which was then added with Griess's reagents of 1% sulfanilamide in 5% phosphoric acid and 0.1% NED (N-1-naphthylethylenediamine dihydrochloride in deionized water.This was then incubated in the dark for 10 min, which was followed by measuring the absorbance at 540 nm using a BioTek Synergy H1 plate reader (BioTek Instruments, Winooski, VT, USA).A standard curve was obtained by using a 100 µM solution of sodium nitrite to quantify the concentration of nitrite.

Cytotoxicity Measurement
The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay was employed to analyze the toxicity of the microparticulate DPD analogs toward murine dendritic cells (DC 2.4) [42] DC 2.4 cells (2.5 × 10 5 cells/well, in 96-well plate) were placed at various concentrations of microparticulate DPD analogs for 48 h at 37 • C. At the end of 48 h, MTT reagent (5 µg/mL in PBS) was added to the supernatants from all the wells and incubated at 37 • C for 4 h, protected from light.The precipitate formazan was then dissolved by the addition of DMSO (dimethyl sulfoxide) and shaking the plate for 15 min at room temperature, protected from light.At the end of 15 min, the absorbance was quantified using a BioTek Synergy H1 plate reader (BIO-TEK Instruments, Winooski, VT, USA) at 570 nm.

In Vitro Release Study
An in vitro release study of C8-HSL was conducted to determine the percentage of DPD analogs released from the PLGA polymer [43].Briefly, 5 mg of each analog of DPD (ent-DPD, n-butyl-DPD, isobutyl-DPD, n-hexyl-DPD, phenyl-DPD) MP was weighed in a balance, which was then added to Eppendorf tubes, where each tube contained 1 mL of phosphate buffered saline (PBS).Each of the Eppendorf tubes was then placed in an incubator at 60 rpm at 37 • C temperature.The supernatants were collected and replaced with PBS at 0, 1, 2, 3, to 168 h.The sample was then centrifuged at 1500 rpm for 10 min.After completing the centrifuge, the small molecule content that was released was determined via a spectrophotometer (UV) at different wavelengths for the following analogs: ent-DPD (230 nm), n-butyl-DPD (234 nm), isobutyl-DPD (240 nm), n-hexyl-DPD (242 nm), and phenyl-DPD (245 nm) (Thermo-Fisher, Waltham, MA, USA, NANODROP 2000c).

Expression of Antigen-Presenting Molecules
Dendritic cells (DC 2.4) were exposed to different microparticulate formulations.The level of major histocompatibility complexes I and II was quantified.Dendritic cells in population concentration of 50 × 10 4 cells/well in a 48-well plate were exposed to different groups such as marketed measles vaccine (positive control), blank microparticles, ent DPD microparticles, n-butyl-DPD microparticles, isobutyl-DPD microparticles, nhexyl-DPD microparticles, and phenyl-DPD microparticles groups which was then (n = 3) incubated at 37 • C for 48 h.After completion of incubation, the cells were stained with allophycocyanin (APC1) and fluorescein isothiocyanate-labeled MHC I and MHC II markers (eBioscience Laboratories, San Diego, CA, USA) for 1 h at 4 • C, protected from light.The fluorescence intensity was quantified using a BD Accuri C6 plus flow cytometer (BD Bioscience, San Jose, CA, USA).

Expression of Costimulatory Molecules
Antigen-presenting dendritic cells (DC 2.4) were exposed to the different groups of DPD-analogs microparticulate formulations.The level of CD40 and CD80 (costimulatory molecules) was quantified.Dendritic cells with population density of 50 × 10 4 cells/well in a 48-well plate were exposed to different groups, including marketed measles vaccine (positive control), blank microparticles, ent-DPD microparticles, n-butyl-DPD microparticles, isobutyl-DPD microparticles, n-hexyl-DPD microparticles, and phenyl-DPD microparticles (n = 3) groups, which were then incubated at 37 • C for 48 h.After completion of incubation, the cells were stained with allophycocyanin (APC1) and fluorescein isothiocyanate-labeled CD40 and CD80 markers (eBioscience Laboratories, San Diego, CA, USA) for 1 h at 4 • C, protected from light.The fluorescence intensity was measured using a BD Accuri C6 plus flow cytometer (BD Bioscience, San Jose, CA, USA).

Griess's Assay
The efficacy of microparticles as adjuvants was analyzed by quantifying the nitric oxide released from dendritic cells when exposed to different DPD analog microparticulate formulations integrated with gonorrhea vaccine microparticles [27].Nitrite, an oxidation product of nitric oxide, was quantified to evaluate the release of nitric oxide from dendritic cells (DC 2.4).Dendritic 2.4 cells with populations of 50 × 10 4 cells/well in a 48-well plate were exposed to different DPD analog groups, including alum + MF59 ® microparticles, ent-DPD microparticles, n-butyl-DPD microparticles, isobutyl-DPD microparticles, n-hexyl-DPD microparticles, and phenyl-DPD microparticles, combined with gonorrhea vaccine microparticles for 48 h.The concentration of nitrite was then quantified as described in Section 4.2.5.

Adjuvant Effect: Expression of Antigen-Presenting Molecules
Various microparticulate DPD analogs were combined with a microparticulate gonorrhea vaccine and exposed to dendritic cells (DC 2.4) to evaluate the adjuvant effect by assessing the expression of major histocompatibility complex I and II, antigen-presenting molecules.DC 2.4 cells (50 × 10 4 cells/well in a 48-well plate) were exposed to the combination of various microparticulate DPD analogs and microparticulate formulations of FDA-approved adjuvants (alum and AddaVax TM ) with gonorrhea vaccine microparticles (n = 3), which were then incubated at 37 • C temperature for 48 h.Major histocompatibility complex I and II expression on the surface of the antigen-presenting dendritic cells (DC 2.4) was then quantified as described in Section 4.2.8..

Adjuvant Effect: Expression of Costimulatory Molecules
Various microparticulate DPD analogs were combined with a microparticulate gonorrhea vaccine and exposed to dendritic cells (DC 2.4) to evaluate the adjuvant effect by assessing the expression of CD40 and CD80, costimulatory molecules.DC 2.4 cells (50 × 10 4 cells/well in a 48-well plate) were exposed to the combination of various mi-croparticulate DPD analogs and microparticulate formulations of FDA-approved adjuvants (alum and AddaVax TM ) with gonorrhea vaccine microparticles (n = 3), which were then incubated at 37 • C temperature for 48 h.The expression of costimulatory molecules CD40 and CD80 on the surface of the antigen-presenting dendritic cells (DC 2.4) was then evaluated as described in Section 4.2.9.

Statistical Analysis
All experiments were replicated three times.GraphPad Prism 5 software was used for statistical analyses.The results are represented as the mean ± SEM.An unpaired two-tailed t test was used for comparison of different experimental groups.A one-way ANOVA followed by Tukey's post hoc analysis was used for comparison of multiple groups.In this study, all the experiments were conducted in triplicate unless otherwise stated.A Shapiro-Wilk test was used to test the normality of the data and the Brown Forsythe test was used to evaluate the equality of variances.A one-way analysis of variance (ANOVA) with Tukey's post hoc test was performed for datasets with normal distribution.

Figure 2 .
Figure 2. The whole-cell inactivated N. gonorrhea was encapsulated into a polymer matrix to form vaccine microparticles (MP).

Figure 4 .
Figure 4. Level of nitric oxide released from dendritic cells was measured in Griess's assay.Murin dendritic cells (DC2.4) (5 × 10 5 cells) were pulsed with different groups of microparticles (dose 5 µg/5 × 10 5 cells) for 48 h.Nitrite level in supernatant was measured using Griess's reagents.The was no significant difference between nitric oxide released from marketed measles vaccine com pared to ent-DPDMPs, n-butyl-DPD MPs, and isobutyl-DPD MPs.However, cells exposed marketed measles vaccine released remarkably higher amounts of nitric oxide than the groups cells exposed to n-hexyl-DPD MPs and phenyl-DPD MPs.Results are presented as the mean SEM (n = 3).*** p < 0.001 extremely significant.

Figure 5 .Figure 4 .
Figure 5. DPD analogs microparticles ((A) ent-DPD MPs, (B) n-butyl-DPD MPs, (C) isobutyl-DPD MPs, (D) n-hexyl-DPD MPs, and (E) phenyl-DPD MPs) were found to be noncytotoxic dendritic cells (DCs).DCs were pulsed with increasing doses of microparticulate DPD analogs an incubated for 48 h.The microparticles were not cytotoxic compared to dimethyl sulfoxide (DMSO Here, we compared the cells only group versus the lowest concertation, which was 50 ug/mL.Th indicates that the lowest concentration is safe to use as a dose using DCs.In addition, the cells we only used to compare them versus other concentrations.Cytotoxicity of each analog was analyze

Figure 4 .
Figure 4. Level of nitric oxide released from dendritic cells was measured in Griess's assay.Murine dendritic cells (DC2.4) (5 × 10 5 cells) were pulsed with different groups of microparticles (dose 50 µg/5 × 10 5 cells) for 48 h.Nitrite level in supernatant was measured using Griess's reagents.There was no significant difference between nitric oxide released from marketed measles vaccine compared to ent-DPDMPs, n-butyl-DPD MPs, and isobutyl-DPD MPs.However, cells exposed to marketed measles vaccine released remarkably higher amounts of nitric oxide than the groups of cells exposed to n-hexyl-DPD MPs and phenyl-DPD MPs.Results are presented as the mean ± SEM (n = 3).*** p < 0.001 extremely significant.

Figure 5 .Figure 5 .
Figure 5. DPD analogs microparticles ((A) ent-DPD MPs, (B) n-butyl-DPD MPs, (C) isobutyl-DPD MPs, (D) n-hexyl-DPD MPs, and (E) phenyl-DPD MPs) were found to be noncytotoxic in dendritic cells (DCs).DCs were pulsed with increasing doses of microparticulate DPD analogs and incubated for 48 h.The microparticles were not cytotoxic compared to dimethyl sulfoxide (DMSO).Here, we compared the cells only group versus the lowest concertation, which was 50 ug/mL.This indicates that the lowest concentration is safe to use as a dose using DCs.In addition, the cells were only used to compare them versus other concentrations.Cytotoxicity of each analog was analyzed

Figure 8 .
Figure 8. Costimulatory molecules CD40 and CD80.Level of (A) CD40 and (B) CD80 on the surface of murine dendritic cells.Results are expressed as mean ± SEM (n = 3).Dendritic cells showed significantly lower expression upon exposure to the n-hexyl-DPD microparticles and phenyl-DPD microparticles compared to the positive control.The term "There was no significant difference between" was used to compare the different groups based on the results of the statistical test.In Figure

2. 11 . 1 .
Evaluation of Adjuvant Effect with MenAfriVac (MAV), a Meningococcal A Conjugate Vaccine Microparticle The following figure shows the adjuvant effect of MenAfriVac (MAV), Meningococcal A Conjugate Vaccine Microparticle with all five types of DPD Analogs.

2. 11 . 1 .
Evaluation of Adjuvant Effect with MenAfriVac (MAV), a Meningococcal A Conjugate Vaccine Microparticle The following figure shows the adjuvant effect of MenAfriVac (MAV), Meningococcal A Conjugate Vaccine Microparticle with all five types of DPD Analogs.

Pharmaceuticals 2023 ,
16, x FOR PEER REVIEW 15 of 25The following figure shows the adjuvant effect of Influenza Vaccine combined with all five types of DPD Analogs.

Figure 13 .
Figure 13.Influenza (S) vaccine was combined with ent-DPD, n-butyl-DPD, isobutyl-DPD MPs, n-hexyl-DPD MPs, and phenyl-DPD MPs.Release of nitric oxide from dendritic cells as calculated from Griess's assay.Results are expressed as mean ± SEM (n = 3).*** p < 0.001 extremely significant.2.11.5.Evaluation of adjuvant effect with inactivated Zika vaccine microparticlesThe following figure shows the adjuvant effect of Zika Vaccine combined with all five types of DPD Analogs.

Figure 13 .
Figure 13.Influenza (S) vaccine was combined with ent-DPD, n-butyl-DPD, isobutyl-DPD MPs, n-hexyl-DPD MPs, and phenyl-DPD MPs.Release of nitric oxide from dendritic cells as calculated from Griess's assay.Results are expressed as mean ± SEM (n = 3).*** p < 0.001 extremely significant.2.11.5.Evaluation of adjuvant effect with inactivated Zika vaccine microparticlesThe following figure shows the adjuvant effect of Zika Vaccine combined with all five types of DPD Analogs.

Figure 17 .
Figure 17.Schematic representation of the method for formulation of microparticulate DPD analogs.

Figure 17 .
Figure 17.Schematic representation of the method for formulation of microparticulate DPD analogs.
percent recovery yield = Weight o f microparticles × 100 Weight o f all ingredients in the f ormulation (1)

Table 2 .
Formulation and characterization of microparticulate DPD analogs.Values in parentheses were measured before lyophilization.