Dose-Dependent Prophylactic Efficacy of Filarial Antigens Glutathione-S-Transferase and Abundant Larval Transcript-2 against Brugia malayi Challenge in Mastomys

Objective To identify the most effective dose of filarial rBmALT-2 and rWbGST alone or in combination against B. malayi infection in vitro and in vivo. Methods Mastomys (n = 5–7/group) received intramuscular (i.m.) injection with three different doses (25, 50, and 100 μg) of rBmALT-2 or rWbGST, either alone or in combination with alum as the adjuvant. Protective immunity was studied by in vivo and in vitro cytotoxicity assay. To evaluate the cellular immune response, splenocyte proliferation and cytokine profile were assessed. Results Serological results revealed a substantial (p < 0.005) induction of IgG1, IgG2a, and IgG3 responses in vaccinated Mastomys. Mastomys immunized with 50 μg rBmALT-2 + alum induced 79–81% killing against the L3 larvae challenge in vivo and in vitro ADCC assay (p < 0.005); whereas rWbGST + alum alone or in combination with rBmALT-2 + alum induced 63–68% killing (p < 0.005) in vivo and in vitro. Antigen-specific cytokine profiles of Mastomys vaccinated with either BmALT-2, WbGST or a combination showed elevated IL-10, IL-4, and IFN-γ levels, signifying both Th1 and Th2 immune response. Conclusions These findings suggest that immunization of Mastomys with a 50 μg/dose of rBmALT-2 + alum four times at a 4-week interval demonstrated considerable protection against B. malayi infection.


Introduction
Parasitic worms, specifcally Wuchereria bancrofti, Brugia malayi, and B. timori, cause lymphatic flariasis (LF), infuencing roughly 863 million individuals worldwide [1].Mass drug administration (MDA) is one of the strategies used by the World Health Organization (WHO) in the Global Programme to Eliminate Lymphatic Filariasis, which aims to eradicate the disease by 2030 [1].Since consistent MDA renders parasites vulnerable to developing drug resistance, several treatment modes should be considered.Prophylactic vaccination is one such approach that may aid in LF control and elimination worldwide [2].Moreover, LF re-emergence in several countries has highlighted the importance of developing a prophylactic vaccine for LF [3][4][5].
Tis study aimed to evaluate the efective dose of both antigens (rBmALT-2 and rWbGST) either as a single or combination vaccine in Mastomys coucha.
For cytokine analysis, similar sets of single cell suspension (2 × 10 6 cells/mL/well) were prepared and plated in cell culture plates (24-well plate), which were further stimulated with 10 μg/well of the respective antigens or 2 μg/ well of ConA (positive control) and incubated for 72 h (37 °C in 5% CO 2 ).Te cytokine ELISA kits (Invitrogen Bioservices, Bengaluru, India) were used to assess the amounts of IL-4, IL-10, and IFN-c in the culture supernatants after incubation following the manufacturer's instructions.

Antigen-Specifc IgG Titer and Isotype in the Sera of Vaccinated
Mastomys.An indirect ELISA was used to quantify the amounts of rBmALT-2-or rWbGST-specifc total IgG antibodies in the sera of Mastomys [11].Te serum samples collected on day 100 (Figure 1) were diluted at dilutions of 1 : 100, 1 : 500, 1 : 1000, and 1 : 10000 and tested for the presence of IgG antibodies using a goat anti-mouse HRPconjugated secondary antibody diluted 1 : 10000.(Termo Fisher Scientifc, Mumbai, India).TMB substrate was used to develop the reaction, and adding 2M H 2 SO 4 stopped the color development.A spectrophotometer (Biotek, New Delhi) was used to measure the absorbance at 450 nm.
Te concentrations of antigen-specifc isotypes (such as IgG1, IgG2a, IgG2b, and IgG3) against rBmALT-2 or rWbGST were measured in the blood samples using an indirect ELISA method, as explained in a previous study [11], with the use of the specifc HRP-labeled antibodies for each isotype.

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Canadian Journal of Infectious Diseases and Medical Microbiology

Statistical Assessment.
Te statistical studies were conducted using SPSS V21.0 software (IBM, SPSS Inc., India).Survival data were examined using one-way analysis of variance (ANOVA) with a Bonferroni post hoc test.p values <0.05 were considered signifcant.Sera and cytokine were analyzed by the Kruskal-Wallis test followed by Bonferroni post-test multiple comparisons, p values <0.05 and < 0.005 were considered signifcant.

ADCC Assay Showed Larvicidal Activity by Antigen-Specifc Antibodies in the Sera of Immunized Mastomys.
Te serum of immunized Mastomys, with diferent doses, 25, 50, or 100 μg rBmALT-2, rWbGST or their combination, promoted PEC adherence to the L3.All sera of the immunized groups showed signifcantly higher ADCC than that of the control group; 50 μg rBmALT-2 induced the highest protection (Table 1).Furthermore, regardless of the antigen dose, only Mastomys sera immunized with rBmALT-2 had the highest activity against L3 (p < 0.005) compared to other test antigens.

. Discussion
Te present work focuses on two well-documented and potent vaccine candidates: rBmALT-2 and rWbGST.Previous studies showed that immunization with these proteins individually can induce signifcant (p < 0.05) protection against flariasis in Jirds and Mastomys [6,7,18].Antigen immunogenicity is infuenced not only by its physical and biological activities but also by its dose [23].Several fndings have suggested that a modest amount of immunogen may produce cell-mediated immunity (CMI), whereas high doses may elicit humoral immune responses [24][25][26].However, some research groups indicate that the type and dose of immunogen utilized mostly regulates the immune response [27,28].Hence, this study used purifed rBmALT-2 and rWbGST at diferent doses, 25, 50, and 100 μg, since administering large doses may induce a systemic immune response [29].
Using an in vitro and in vivo, ADCC and difusion chamber, approach for this investigation, we found higher protective efcacy with 50 μg rBmALT-2 than with 25 and 100 μg rBmALT-2, all doses of rWbGST or the combinations of rBmALT-2 + rWbGST when alum was used as an adjuvant against B. malayi L3.Combination of these two antigens induced approximately 64-68% protection against B. malayi L3 in vitro [2,10,30,31].In our study, rBmALT-2 induced the highest protective efcacy (69-80% in vitro) regardless of the dose administered.
Te in vivo difusion chamber approach, which is performed in a limited physiological environment, has been utilized to investigate parasitic survival, development, and the host efector mechanism.Some experiments using B. malayi have been used to show comparable results between in vitro and in vivo assays [11,36].Macrophage participation in cytotoxicity has been verifed against different flarial parasites, such as Dipitelonema setariosum adults [37] and the L3 of Litomosoides sigmodontis [38].In the current study, we discovered that the in vivo difusion chamber cytotoxicity results were consistent with those obtained from the in vitro ADCC test.Te in vivo, difusion chamber experiment showed 78%, 59%, and 65% protective efcacy with 50 μg rBmALT-2, rWbGST, and their combination, respectively.Similarly, Mastomys immunized with 25 or 100 μg rBmALT-2 showed higher larval killing (approximately 68-73%) than rWbGST or the combination rBmALT-2 + rWbGST (61-65%).Mastomys immunized intramuscularly with 50 μg rBmALT-2 showed better protective efcacy than other immunogens, both in vitro and in vivo.Recombinant BmALT-2 is one of the flarial antigens that can provide substantial protection against L3 [8,36,39,40].Although the exact mechanism of rBmALT-2 protein is unknown, studies indicate that it may be crucial for host immunomodulation [38,41].
Notably, the rBmALT-2 + rWbGST combination showed higher and lower protective efcacy (65%) than rWbGST alone (61%) and rBmALT-2 alone (73%) in vivo.Tis study does not rule out the potential that an instantaneous association among these two antigens (rBmALT-2 and rWbGST) may reduce rBmALT-2 immunogenicity.rWbGST may have concealed the antigenic epitopes on rBmALT-2, reducing rBmALT-2 efcacy in conjugation vaccination [30].A study on malaria examined the protective potential of two recombinant Plasmodium yoelli merozoite surface proteins (MSPs), namely, PyMSP-8 and PyMSP-142, which has shown a similar result.When mice were treated with a combination of these antigens, the Canadian Journal of Infectious Diseases and Medical Microbiology immunogenicity of PyMSP-142 decreased [42].In a flarial study Jirds, immunization by conjugating rBmTGA and rBmALT-2 showed merely 47% protection against B. malayi L3 [30].Terefore, selecting antigens for a successful combination is essential, as not every combination yields a positive outcome.
Te T-cell response, typically associated with elevated antibody levels, was investigated using IgG isotypes and cytokine profles.Te result indicated that Mastomys immunized with 50 or 100 μg rWbGST or rBmALT-2 or combination of both antigens (rBmALT-2 + rWbGST) had signifcant levels of IgG1, IgG2a, and IgG3 antibodies, indicating T1 and T2-type reaction.IgG1 and IgG2a isotypes in mice are involved in complement fxation and binding to protein antigens, while IgG3 is involved in carbohydratecontaining epitope identifcation [36,41].Mouse immune complexes with IgG2a and IgG3 can attach to FccRI and initiate receptor-mediated responses [36,43].Tese isotypes also contribute to the immune response of vaccines and the in vitro reactions (ADCC) against invading pathogens.[44,45].Our fndings indicate that vaccine candidates and their combination elicited mixed T1/T2 immune responses.Some research fndings suggest that T2 immune responses contribute to intestinal helminth resistance [45], although applying these fndings to tissue-dwelling nematodes, like LF [45], is challenging.Te T1-type immune response is essential to stimulate immune protection against flarial infection [45].Intramuscular immunization with 50 μg rBmALT-2 with alum against flarial infection is associated with elevated IFN-c, IL-4, and IL-10 levels and protective T1 and T2 immune responses.
In conclusion, Mastomys immunized four times at a 4week interval with 50 μg rBmALT-2 plus alum as the adjuvant showed an enhanced total IgG antibody titer levels.Te isotype profle showed increases in IgG2a, followed by IgG3 and IgG1 levels, demonstrating a mixed T1 and T2 response.Immunization with rBmALT-2 plus alum also signifcantly increased levels of IFN-c, IL-4, and IL-10 in the spleen cells stimulated ex vivo., indicating a mixed T1 and T2 response.Moreover, the immunization presented higher protection of approximately 80% against L3 larvae in in vitro ADCC and in vivo difusion chamber assay.Terefore, 50 μg/intramuscular dose of rBmALT-2 plus alum as the adjuvant can be used as an efective immunization dose for human LF to explore further preclinical and mechanistic studies.Canadian Journal of Infectious Diseases and Medical Microbiology

Figure 4 :Figure 5 :
Figure 4: Percent protection in immunized Mastomys was calculated by determining the percent larval death.(a) Mastomys were immunized i.m. at 4 weeks of intervals with diferent doses (25, 50, and 100 μg) of rBmALT-2, rWbGST, rBmALT-2 + rWbGST, and alum.Ten days after the last immunization, 20 live L3 of B. malayi placed in a micropore chamber were surgically implanted into the peritoneal cavity of each Mastomys.Chambers were removed aseptically after 48 h, and the L3 death was determined.(b) Several cells were found attached to the dead larvae in the vaccinated Mastomys.However, no cells were attached to the live larvae collected from Mastomys immunized with alum only.Each bar represents the mean ± SD, n � 5-7 per group.* p < 0.05, * * p < 0.005 compared to control-alum group as analyzed by Kruskal-Wallis test, followed by the Bonferroni post hoc test multiple comparison test.

Figure 6 :
Figure6: Levels of secreted cytokines (IFN-c, IL-4, and IL-10) in the culture supernatants of spleen cells stimulated for 72 h at 37 °C in 5% CO 2 with (a) rBmALT-2 or (b) rWbGST were measured using an ELISA.Te concentration of each cytokine represented as pg/mL; bar represents the mean ± SD, n � 5-7 per group.* p < 0.05, * * p < 0.005 compared to control-alum group as analyzed by Kruskal-Wallis test, followed by the Bonferroni post hoc test multiple comparison test.

Table 1 :
(25,itro ADCC assay.B. malayi L3 were incubated for 48 h with normal peritoneal exudate (PECs) cells and with the pooled sera (50 μL) of immunized(25, 50, or 100 μg rBmALT-2 or rWbGST or their combination) or control group of the Mastomys.Te percentages of protection were calculated from total, live, and dead larvae.Data represent the mean ± SD from diferent sets of experiments (in duplicates), n � 5-7/groups; the Kruskal-Wallis test and the Bonferroni multiple comparisons test were used to examine the data, compared to the control-alum group.Te percentages of protection were calculated from total, live, and dead larvae.Data represent the mean ± SD from diferent sets of experiments (in duplicates), n � 5-7/groups; the Kruskal-Wallis test and the Bonferroni multiple comparisons test were used to examine the data.* p < 0.05, * * p < 0.005 compared to the control-alum group.