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. 2016 Apr 5;10(4):e0004586.
doi: 10.1371/journal.pntd.0004586. eCollection 2016 Apr.

Vaccination of Gerbils with Bm-103 and Bm-RAL-2 Concurrently or as a Fusion Protein Confers Consistent and Improved Protection against Brugia malayi Infection

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Vaccination of Gerbils with Bm-103 and Bm-RAL-2 Concurrently or as a Fusion Protein Confers Consistent and Improved Protection against Brugia malayi Infection

Sridhar Arumugam et al. PLoS Negl Trop Dis. .

Abstract

Background: The Brugia malayi Bm-103 and Bm-RAL-2 proteins are orthologous to Onchocerca volvulus Ov-103 and Ov-RAL-2, and which were selected as the best candidates for the development of an O. volvulus vaccine. The B. malayi gerbil model was used to confirm the efficacy of these Ov vaccine candidates on adult worms and to determine whether their combination is more efficacious.

Methodology and principle findings: Vaccine efficacy of recombinant Bm-103 and Bm-RAL-2 administered individually, concurrently or as a fusion protein were tested in gerbils using alum as adjuvant. Vaccination with Bm-103 resulted in worm reductions of 39%, 34% and 22% on 42, 120 and 150 days post infection (dpi), respectively, and vaccination with Bm-RAL-2 resulted in worm reductions of 42%, 22% and 46% on 42, 120 and 150 dpi, respectively. Vaccination with a fusion protein comprised of Bm-103 and Bm-RAL-2 resulted in improved efficacy with significant reduction of worm burden of 51% and 49% at 90 dpi, as did the concurrent vaccination with Bm-103 and Bm-RAL-2, with worm reduction of 61% and 56% at 90 dpi. Vaccination with Bm-103 and Bm-RAL-2 as a fusion protein or concurrently not only induced a significant worm reduction of 61% and 42%, respectively, at 150 dpi, but also significantly reduced the fecundity of female worms as determined by embryograms. Elevated levels of antigen-specific IgG were observed in all vaccinated gerbils. Serum from gerbils vaccinated with Bm-103 and Bm-RAL-2 individually, concurrently or as a fusion protein killed third stage larvae in vitro when combined with peritoneal exudate cells.

Conclusion: Although vaccination with Bm-103 and Bm-RAL-2 individually conferred protection against B. malayi infection in gerbils, a more consistent and enhanced protection was induced by vaccination with Bm-103 and Bm-RAL-2 fusion protein and when they were used concurrently. Further characterization and optimization of these filarial vaccines are warranted.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. SDS-PAGE of purified Brugia malayi recombinant proteins, Bm-RAL-2, Bm-103 and Bm-RAL-2—Bm-103 fusion protein.
Two μg of each protein was loaded in SDS-PAGE along with SeeBlue pre-stained protein marker (Invitrogen, USA). (A) Bm-RAL-2; (B) Bm-103; (C) Bm-RAL-2—Bm-103 fusion protein.
Fig 2
Fig 2. Effect of vaccination with Bm-RAL-2 and Bm-103 individually, concurrently and as a fusion protein on the development of protective immunity manifested as % reduction in worm burdens and % host protection against infection with Brugia malayi L3s in Mongolian gerbils.
(A) Vaccination with Bm-RAL-2 (42 dpi); (B) Vaccination with Bm-103 (42 dpi); (C) Vaccination with Bm-RAL-2—Bm-103 fusion protein or with Bm-RAL-2 + Bm-103 concurrently (90 dpi); (D) Vaccination with Bm-RAL-2—Bm-103 fusion protein or Bm-RAL-2 + Bm-103 concurrently (150 dpi). Each dot represents the total number of adult worms recovered from an individual animal. The lines represent mean with standard deviation. P ≤ 0.05 denotes a statistically significant difference in total worm recovery (percent reduction) between vaccinated and alum control group, Mann–Whitney U Test, GraphPad Prism 6.
Fig 3
Fig 3. Total IgG endpoint titers specific to Bm-103, Bm-RAL-2 or the fusion protein in gerbils vaccinated (N = 10) with Bm-RAL-2 and Bm-103 individually, concurrently or as a fusion protein.
Each dot represents IgG endpoint titer from an individual animal. The lines represent median with maximum and minimum range. Asterisk/s denotes a statistically significant difference in IgG endpoint titers between analyzed groups, Mann–Whitney U Test, GraphPad Prism 6 (*, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001).
Fig 4
Fig 4. Embryogram data of B. malayi female worms recovered 150 dpi from alum control, Bm-RAL-2 and Bm-103 vaccinated gerbils.
(A) Eggs/female worm; (B) Embryos/female worm; (C) Total embryonic stages/female worm. Statistical significance was determined by Mann–Whitney U Test using GraphPad Prism version 6, P ≤ 0.05. The line represents mean with standard deviation.
Fig 5
Fig 5. Embryogram data of B. malayi female worms recovered 150 dpi from alum control, Bm-103 + Bm-RAL-2 and Bm-103—Bm-RAL-2 vaccinated gerbils.
(A) Eggs/female worm; (B) Embryos/female worm; (C) Total embryonic stages/female worm. Statistical significance was determined by Mann–Whitney U Test using GraphPad Prism version 6, asterisks denotes a significant difference between the alum control and the vaccinated group, P ≤ 0.05. The line represents mean with standard deviation.

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