A Recombinant Trivalent Fusion Protein F1-LcrV-HSP70(II) Augments Humoral and Cellular Immune Responses and Imparts Full Protection against Yersinia pestis

Abstract

Plague is one of the most dangerous infections in humans caused by Yersinia pestis, a Gram-negative bacterium. Despite of an overwhelming research success, no ideal vaccine against plague is available yet. It is well established that F1/LcrV based vaccine requires a strong cellular immune response for complete protection against plague. In our earlier study, we demonstrated that HSP70(II) of Mycobacterium tuberculosis modulates the humoral and cellular immunity of F1/LcrV vaccine candidates individually as well as in combinations in a mouse model. Here, we made two recombinant constructs caf1-lcrV and caf1-lcrV-hsp70(II). The caf1 and lcrV genes of Y. pestis and hsp70 domain II of M. tuberculosis were amplified by polymerase chain reaction. Both the recombinant constructs caf1-lcrV and caf1-lcrV-hsp70(II) were cloned in pET28a vector and expressed in Escherichia coli. The recombinant fusion proteins F1-LcrV and F1-LcrV-HSP70(II) were purified using Ni-NTA columns and formulated with alum to evaluate the humoral and cell mediated immune responses in mice. The protective efficacies of F1-LcrV and F1-LcrV-HSP70(II) were determined following challenge of immunized mice with 100 LD50 of Y. pestis through intraperitoneal route. Significant differences were noticed in the titers of IgG and it's isotypes, i.e., IgG1, IgG2b, and IgG3 in anti- F1-LcrV-HSP70(II) sera in comparison to anti-F1-LcrV sera. Similarly, significant differences were also noticed in the expression levels of IL-2, IFN-γ and TNF-α in splenocytes of F1-LcrV-HSP(II) immunized mice in comparison to F1-LcrV. Both F1-LcrV and F1-LcrV-HSP70(II) provided 100% protection. Our research findings suggest that F1-LcrV fused with HSP70 domain II of M. tuberculosis significantly enhanced the humoral and cellular immune responses in mouse model.

Keywords: F1–LcrV; F1–LcrV–HSP70(II); HSP70(II); Yersinia pestis; cytokine; plague.

FIGURE 1

(A) PCR amplification of caf1 and lcrV genes of Yersinia pestis & cloning of caf1–lcrV in pET vector. caf1 gene (a); Lane 1, 1Kb DNA ladder; Lane 2, caf1 gene product of 513 bp. lcrV gene (b); Lane 1, 1Kb DNA ladder; Lane 2, lcrV gene product of 981 bp. Screening of positive clones of caf1–lcrV fusion product after ligation in pET28a vector (c); Lane 1, 1 Kb DNA ladder; Lane 2–4, released inserts (caf1–lcrV) of ~1500 bp after restriction digestion from pET vector. (B) Recombinant F1–LcrV fusion protein expression profile and purification. (a) SDS–PAGE analysis of F1–LcrV protein expression. Lane 1, Pre-stained protein marker; Lane 2, Uninduced Escherichia coli cell lysate clone; Lane 3, Induced E. coli cell lysate (b) Western blot analysis of F1–LcrV protein showing reaction with anti HIS antibody. Lane 1, Pre-stained protein marker; Lane 2, Uninduced E. coli cell lysate clone; Lane 3, Induced E. coli cell lysate. (c) Purification of F1–LcrV protein. Lane 1, Protein marker; Lane 2, Cell lysate; Lane 3, Flow through; Lane 4, Wash; Lane 5–10, Eluted fractions of protein showing F1–LcrV protein of 54 kDa.

FIGURE 2

(A) PCR amplification of caf1, lcrV genes of Y. pestis and hsp70(II) of Mycobacterium tuberculosis & cloning of caf1–lcrV–hsp70(II) in pET vector. caf1 gene (a); Lane 1, 1 Kb DNA ladder; Lane 2, caf1 gene product of 513 bp: lcrV gene (b); Lane 1, 1 Kb DNA ladder; Lane 2, lcrV gene product of 981 bp: hsp70(II) gene (c); Lane 1, 1 Kb DNA ladder; Lane 2, hsp70(II) gene product of 630 bp. Screening of positive clones of caf1–lcrV–hsp70(II) fusion product after ligation in pET28a vector (d); Lane 1, 1 Kb DNA ladder; Lane 2–4, released inserts (caf1–lcrV) of 2124 bp after restriction digestion from pET vector. (B) Recombinant F1–LcrV-HSP70(II) trivalent fusion protein expression profile & purification. (a) SDS–PAGE analysis of F1–LcrV–HSP70(II) protein expression: Lane 1, Protein marker; Lane 2, Uninduced E. coli cell lysate clone; Lane 3, Induced E. coli cell lysate. (b) Purification of F1–LcrV–HSP70(II) protein: Lane 1, Protein markers; Lane 2, Cell lysate; Lane 3, Flow through; Lane 4, Wash; Lane 5–10, Eluted fractions of purified protein showing F1–LcrV–HSP70(II) fusion protein of 79 kDa. (c) Western blot analysis of purified F1–LcrV–HSP70(II) protein showing reaction with anti HIS antibody: Lane 1, Pre-stained protein marker; Lane 2, purified F1–LcrV–HSP70(II) protein.

FIGURE 3

Representation of animal groups and the schedule of vaccination activities. (A) Prepared groups of Balb/C mice (8/group) for vaccination studies with F1–LcrV and F1–LcrV–HSP70(II) in formulation with alum. (B) Schematic representation of vaccination schedule, blood collection for humoral and cell mediated studies and challenge experiments.

FIGURE 4

End point titers of IgG antibody and IgG isotypes in the sera of immunized mice. (A) IgG end point titers were estimated by ELISA. Serum samples were collected after first and second boosters from vaccinated animal groups, i.e., F1–LcrV and F1–LcrV–HSP70(II)). (B) The IgG isotypes; IgG1, IgG2a, IgG2b, and IgG3 were measured by ELISA. End point titers of IgG isotypes in sera samples collected after second booster from vaccinated mice with F1–LcrV and F1–LcrV–HSP70(II) antigens. Analysis was done by one way ANOVA, all Pairwise Multiple Comparison Procedure (Fisher LSD Method). ** P < 0.01; *** P < 0.001; ^#P < 0.001.

FIGURE 5

Estimation of cytokines in vaccinated animal groups. Splenocytes isolated from immunized BALB/c mice with F1–LcrV and F1–LcrV–HSP70(II) including control group. Splenocytes were stimulated with F1–LcrV and F1–LcrV–HSP70(II) antigens (5 μg/ml each) and after 48 h, the expression levels of cytokines were measured. The expression levels of cytokines (A) IL-2, (B) IFN-γ, and (C) TNF-α were calculated in picograms per milliliter (pg/ml) as shown in graphs. Data represent the average of 8 mice/group ± SD of the results determined. One way ANOVA was applied for analysis. *** P < 0.001; ^#P < 0.001.

FIGURE 6

Vaccination with F1–LcrV and F1–LcrV–HSP70(II) protects mice against Y. pestis (S1 strain). The vaccinated and control group animals were experimentally infected via i.p. route with 100 LD₅₀ of virulent Y. pestis. The survivals of animals were monitored for 30 days after challenge. The survival curve of both the vaccine candidates F1–LcrV and F1–LcrV–HSP70(II) was determined by Kaplan Meier’s method to calculate percentage survivals (**** P < 0.0001).