Clin Exp Vaccine Res.  2014 Jul;3(2):185-193. 10.7774/cevr.2014.3.2.185.

Cellular immunity survey against urinary tract infection using pVAX/fimH cassette with mammalian and wild type codon usage as a DNA vaccine

• Imani Fooladi AA
• Bagherpour G
• Khoramabadi N
• Fallah Mehrabadi J
• Mahdavi M
• Halabian R
• Amin M
• Izadi Mobarakeh J
• Einollahi

• KMID: 1730623
• DOI: http://doi.org/10.7774/cevr.2014.3.2.185

Abstract

PURPOSE
FimH (the adhesion fragment of type 1 fimbriae) is implicated in uropathogenic Escherichia coli (UPEC) attachment to epithelial cells through interaction with mannose. Recently, some studies have found that UPEC can thrive intracellularly causing recurrent urinary tract infection (UTI). Almost all vaccines have been designed to induce antibodies against UPEC. Yet, the humoral immune response is not potent enough to overcome neither the primary UTI nor recurrent infections. However, DNA vaccines offer the possibility of inducing cell mediated immune responses and may be a promising preventive tool.
MATERIALS AND METHODS
In this study, we employed two different open reading frames within mammalian (mam) and wild type (wt) codons of fimH gene. Optimized fragments were cloned in pVAX-1. Expression of the protein in COS-7 was confirmed by western blot analysis after assessing pVAX/fimH(mam) and pVAX/fimH(wt). The constructs were injected to BALB/c mice at plantar surface of feet followed by electroporation.
RESULTS
The mice immunized with both constructs following booster injection with recombinant FimH showed increased interferon-gamma and interleukin-12 responses significantly higher than non-immunized ones (p<0.05). The immunized mice were challenged with UPEC and then the number of bacteria recovered from the immunized mice was compared with the non-immunized ones. Decreased colony count in immunized mice along with cytokine responses confirmed the promising immune response by the DNA vaccines developed in this study.
CONCLUSION
In conclusion, DNA vaccines of UPEC proteins may confer some levels of protection which can be improved by multiple constructs or boosters.

Keyword

DNA vaccines; Urinary tract infections; FimH; Codon usage optimization

MeSH Terms

Animals
Antibodies
Bacteria
Blotting, Western
Clone Cells
Codon*
DNA*
Electroporation
Epithelial Cells
Foot
Immunity, Cellular*
Immunity, Humoral
Interferon-gamma
Interleukin-12
Mannose
Mice
Open Reading Frames
Urinary Tract Infections*
Uropathogenic Escherichia coli
Vaccines
Vaccines, DNA
Antibodies
Codon
DNA
Interferon-gamma
Interleukin-12
Mannose
Vaccines
Vaccines, DNA

Figure

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  Fig. 1 Stimulation assay. There are significant response in DNA vaccine groups and protein group compared to control groups (pV and phosphate buffered saline [PBS]).

• 

  Fig. 2 Interferon-γ (IFN-γ), interleukin (IL)-12, IL-4, and IL-17 levels in spleen cell cultures of mice after 72 hours of stimulation with recombinant FimH antigen. The cytokine levels were determined using enzyme-linked immunosorbent assay. Data represent the means±standard deviations for triplicate culture of eight animals per group. IFN-γ (A) and IL-12 (B) showed significantly response in DNA vaccine group compare to control groups. IL-4 (C) and IL-17 levels (D) showed no significant response in DNA vaccine group compare to control. PBS, phosphate buffered saline. *p<0.05, **p<0.01, ***p<0.001.

• 

  Fig. 3 Challenge of mice with UPEC 35218. Two weeks after the last immunization, the bladders of mice (n=5) infected with 1×108 CFU of UPEC isolate. The levels of bladder colonization were determined 48 hours after challenge by plating tissue homogenates. Solid lines show median of colonization levels. Significant differences between groups were determined by Kruskal-Wallis analysis (Dunn's multiple comparison test) and are shown by brackets with asterisk. PBS, phosphate buffered saline. *p<0.05 was considered as significant.

• 

  Fig. 4 Microscopic analysis of bladder tissue after hematoxylin and eosin staining. Positively was contained of bacterial colonies that adhesion on bladder epthelial (A and B, ×40), showed as a negative control from unchallenged mouse (C, ×40).

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  Fig. 5 IgA enzyme-linked immunosorbent assay assay. The rate IgA response in DNA vaccine (pV-FimH[mam]/[wt]+boost) groups and protein group was increased. PBS, phosphate buffered saline. ***p < 0.001.

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