close
Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2010 Jul;54(7):2994-3001.
doi: 10.1128/AAC.01492-09. Epub 2010 May 17.

Engineering of Lactobacillus jensenii to secrete RANTES and a CCR5 antagonist analogue as live HIV-1 blockers

Luca Vangelista  1 , Massimiliano Secchi, Xiaowen Liu, Angela Bachi, Letong Jia, Qiang Xu, Paolo Lusso
Affiliations

Engineering of Lactobacillus jensenii to secrete RANTES and a CCR5 antagonist analogue as live HIV-1 blockers

Luca Vangelista et al. Antimicrob Agents Chemother. 2010 Jul.

Abstract

The development of effective microbicides for the prevention of HIV-1 sexual transmission represents a primary goal for the control of AIDS epidemics worldwide. A promising strategy is the use of bacteria belonging to the vaginal microbiota as live microbicides for the topical production of HIV-1 inhibitors. We have engineered a human vaginal isolate of Lactobacillus jensenii to secrete the anti-HIV-1 chemokine RANTES, as well as C1C5 RANTES, a mutated analogue that acts as a CCR5 antagonist and therefore is devoid of proinflammatory activity. Full-length wild-type RANTES and C1C5 RANTES secreted by L. jensenii were purified to homogeneity and shown to adopt a correctly folded conformation. Both RANTES variants were shown to inhibit HIV-1 infection in CD4(+) T cells and macrophages, displaying strong activity against HIV-1 isolates of different genetic subtypes. This work provides proof of principle for the use of L. jensenii-produced C1C5 RANTES to block HIV-1 infection of CD4(+) T cells and macrophages, setting the basis for the development of a live anti-HIV-1 microbicide targeting CCR5 in an antagonistic manner.

PubMed Disclaimer

Figures

FIG. 1.
FIG. 1.
Expression and codon optimization of wt and C1C5 RANTES as secreted molecules in L. jensenii. (A) wt and C1C5 RANTES nonoptimized or codon-optimized (CO) versions were visualized in Western blots using polyclonal rabbit anti-human RANTES antibodies. Proteins were expressed under the p23 or ptsH promoter. Supernatant from L. jensenii transformed with the empty pOsel175 vector and E. coli-purified wt RANTES (standard) were used as controls. (B) Codon-optimized nucleotide sequence for wt (wt-CO) or C1C5 (C1C5-CO) RANTES. Modified nucleotides from native cDNA are in bold. Only codons for residues C1 and C5 (optimized and nonoptimized) are indicated for C1C5 RANTES, since the remaining sequence is identical to that of wt-CO or wt nonoptimized RANTES, respectively.
FIG. 2.
FIG. 2.
Anti-HIV-1 activities of L. jensenii-purified wt and C1C5 RANTES. R5 HIV-1 inhibition was tested by an acute infection assay on the human CD4+ T cell clone PM1. (A and B) Inhibition of laboratory-adapted clade B HIV-1BaL (A) or HIV-1SF162 (B) was tested using L. jensenii-purified wt and C1C5 RANTES and E. coli-purified wt RANTES as a control. (C) Inhibition of primary clade B isolates HIV-15513 and HIV-110005 by L. jensenii-purified wt and C1C5 RANTES. (D) Inhibition of primary clade C isolates HIV-198IN007 and HIV-192BR025 by L. jensenii-purified wt and C1C5 RANTES. HIV-1 inhibition was measured by a p24-based assay after 4 days of infection; values indicate the means ± SD for two independent experiments performed in triplicate.
FIG. 3.
FIG. 3.
Inhibition of HIV-1BaL infection in human MDM. HIV-1 inhibition was tested using L. jensenii-purified wt and C1C5 RANTES. Anti-HIV-1 activity was measured by a p24-based assay after 4 days of infection; values indicate the means ± SD for two independent experiments performed in quadruplicate.
FIG. 4.
FIG. 4.
Molecular characterization of purified wt and C1C5 RANTES secreted by L. jensenii. (A) Mass spectrometry analysis of purified full-length wt RANTES. The deconvoluted mass spectrum is shown, which identifies the major component as the full-length protein and minor components corresponding to a 3-68 fragment and a 1-67 or 2-68 fragment (a serine residue is lacking, and RANTES presents a serine at both termini, positions 1 and 68). (B) The deconvoluted mass spectrum of purified full-length C1C5 RANTES is shown. Arrows connect peaks differing by 16 Da, likely corresponding to different oxidation states of the same protein form (i.e., the 7,877-Da full-length form and the 7,949-Da extra N-terminal alanine form). (C) The proteolytic site of the low-molecular-mass C1C5 RANTES peptide was identified at residue 13 from the N terminus. (D) Western blot analysis of heparin-purified RANTES. Lane 1, RANTES standard; lane 2, RANTES semipurified by ion-exchange chromatography; lane 3, RANTES purified by heparin binding affinity.
See this image and copyright information in PMC

References

    1. Bolton, M., A. van der Straten, and C. R. Cohen. 2008. Probiotics: potential to prevent HIV and sexually transmitted infections in women. Sex. Transm. Dis. 35:214-225. - PubMed
    1. Boyd, M. R., K. R. Gustafson, J. B. McMahon, R. H. Shoemaker, B. R. O'Keefe, T. Mori, R. J. Gulakowski, L. Wu, M. I. Rivera, C. M. Laurencot, M. J. Currens, J. H. Cardellina II, R. W. Buckheit, Jr., P. L. Nara, L. K. Pannell, R. C. Sowder II, and L. E. Henderson. 1997. Discovery of cyanovirin-N, a novel human immunodeficiency virus-inactivating protein that binds viral surface envelope glycoprotein gp120: potential applications to microbicide development. Antimicrob. Agents Chemother. 41:1521-1530. - PMC - PubMed
    1. Chancey, C. J., K. V. Khanna, J. F. Seegers, G. W. Zhang, J. Hildreth, A. Langan, and R. B. Markham. 2006. Lactobacilli-expressed single-chain variable fragment (scFv) specific for intercellular adhesion molecule 1 (ICAM-1) blocks cell-associated HIV-1 transmission across a cervical epithelial monolayer. J. Immunol. 176:5627-5636. - PubMed
    1. Chang, T. L., C. Chang, D. A. Simpson, Q. Xu, P. K. Martin, L. A. Lagenaur, G. K. Schoolnik, D. D. Ho, S. L. Hillier, M. Holodniy, J. A. Lewicki, and P. P. Lee. 2003. Inhibition of HIV infectivity by a natural human isolate of Lactobacillus jensenii engineered to express functional two-domain CD4. Proc. Natl. Acad. Sci. U. S. A. 100:11672-11677. - PMC - PubMed
    1. Cherpes, T. L., M. A. Melan, J. A. Kant, L. A. Cosentino, L. A. Meyn, and S. L. Hillier. 2005. Genital tract shedding of herpes simplex virus type 2 in women: effects of hormonal contraception, bacterial vaginosis, and vaginal group B streptococcus colonization. Clin. Infect. Dis. 40:1422-1428. - PubMed

Publication types