Mycobacterial Heat Shock Protein Hsp65 Research Articles

Serological Testing for Mycobacterial Heat Shock Protein Hsp65 Antibody in Health and Diseases.

Mycobacterial heat shock protein 65 gene (Hsp65) has been widely used for classification of Mycobacterial species, and detection of Mycobacterial genes by molecular methods and has proven useful in identification of Mycobacterial infection in various clinical conditions. Circulating antibody against Mycobacterial hsp65 has been found in many clinical diseases including autoimmune diseases (Crohn’s disease, lupus erythematosus, multiple sclerosis, diabetes, etc.), atherosclerosis and cancers. The prevalence of anti-Hsp65 antibody in the normal healthy population is unknown. We determined the blood levels of antibody against Mycobacterial hsp65 in the normal population represented by 288 blood donors of the American Red Cross and tested the blood of 109 patients with Crohn’s disease and 28 patients with Sjogren’s syndrome for comparison. The seroprevalence of anti-Hsp65 IgG in the normal population of Red Cross donors was 2.8% (8 of 288 positive). The Hsp65 antibody levels were significantly elevated in patients with Crohn’s disease and Sjogren’s syndrome. The prevalence of Hsp65 antibody in Crohn’s disease patients was 67.9% (74 of 109 patients), and 85.7% for Sjogren’s patients (24 of 28 patients). Our data indicate that anti-Hsp65 antibody is rare in the normal population, but frequent in chronic diseases. The presence of circulating Hsp65 antibody reflects an abnormal immune (adaptive) response to Mycobacterial exposure in patients with chronic diseases, thus differentiating the patients with chronic diseases from those clinical mimics.

Dimerization of an Immunoactivating Peptide Derived from Mycobacterial hsp65 Using N-Hydroxysuccinimide Based Bifunctional Reagents Is Critical for Its Antitumor Properties

We have shown previously that a short pentapeptide derived from the mycobacterial heat shock protein hsp65 can be highly activating for the immune system based on its strong reactivity with the early activation antigen of lymphocytes CD69. Here, we investigated an optimal form of presentation of this antigen to the cells of the immune system. Four different forms of the dimerized heptapeptide LELTEGY, and of the control inactive dimerized heptapeptide LELLEGY that both contained an extra UV active glycine-tyrosine sequence, were prepared using dihydroxysuccinimidyl oxalate (DSO), dihydroxysuccinimidyl tartarate (DST), dihydroxysuccinimidyl glutarate (DSG), and dihydroxysuccinimidyl suberate (DSS), respectively. Heptapeptides dimerized through DST and DSG linkers had optimal activity in CD69 precipitation assay. Moreover, dimerization of active heptapeptide resulted in a remarkable increase in its proliferation activity and production of cytokines in vitro. Furthermore, while DST and DSG dimerized heptapeptides both significantly enhanced the cytotoxicity of natural killer cells in vitro, only the DSG dimerized compound was active in suppressing growth of melanoma tumors in mice and in enhancing the cytotoxic activity of tumor infiltrating lymphocytes ex vivo. Thus, while the dimerization of the immunoactive peptide caused a dramatic increase in its immunoactivating properties, its in vivo anticancer properties were influenced by the chemical nature of linker used for its dimerization.

Synthesis of Multivalent Glycoconjugates Containing the Immunoactive LELTE Peptide: Effect of Glycosylation on Cellular Activation and Natural Killing by Human Peripheral Blood Mononuclear Cells

Pentapeptide diacidic sequence LELTE, derived from the mycobacterial heat shock protein hsp65, has been recently identified as a "danger" signal of the immune system effective via specific binding to the universal leukocyte triggering receptor CD69. This sequence is not active per se, only after its presentation within the multivalent environment of its parent protein, or after artificial dimerization using a standard bifunctional reagents. Here we describe an entirely new way of presenting of this peptide based on its attachment to a cyclopeptide RAFT scaffold (K-K-K-P-G)(2) through the epsilon-amino group of lysine residues, alone or in combination with the carbohydrate epitope alphaGalNAc. The ability of such RAFT scaffolds to precipitate the target CD69 receptor or to activate CD69-positive cells is enhanced in compounds 2 and 4 possessing combined peptide/carbohydrate expression. Compounds 2 and 4 are highly efficient activators of natural killer lymphocytes, but they are completely inactive from the point of view of activation-induced apoptosis of lymphocytes by the target cells. These unique properties make the combined peptide/carbohydrate RAFTs highly suitable for future evaluation in animal tumor therapies in vivo and predict them to be readily available and efficient immunoactivators.

Ten tandem repeats of β-hCG 109–118 enhance immunogenicity and anti-tumor effects of β-hCG C-terminal peptide carried by mycobacterial heat-shock protein HSP65

The β-subunit of human chorionic gonadotropin (β-hCG) is secreted by many kinds of tumors and it has been used as an ideal target antigen to develop vaccines against tumors. In view of the low immunogenicity of this self-peptide,we designed a method based on isocaudamer technique to repeat tandemly the 10-residue sequence X of β-hCG (109–118), then 10 tandemly repeated copies of the 10-residue sequence combined with β-hCG C-terminal 37 peptides were fused to mycobacterial heat-shock protein 65 to construct a fusion protein HSP65-X10-βhCGCTP37 as an immunogen. In this study, we examined the effect of the tandem repeats of this 10-residue sequence in eliciting an immune by comparing the immunogenicity and anti-tumor effects of the two immunogens, HSP65-X10-βhCGCTP37 and HSP65-βhCGCTP37 (without the 10 tandem repeats). Immunization of mice with the fusion protein HSP65-X10-βhCGCTP37 elicited much higher levels of specific anti-β-hCG antibodies and more effectively inhibited the growth of Lewis lung carcinoma (LLC) in vivo than with HSP65-βhCGCTP37, which should suggest that HSP65-X10-βhCGCTP37 may be an effective protein vaccine for the treatment of β-hCG-dependent tumors and multiple tandem repeats of a certain epitope are an efficient method to overcome the low immunogenicity of self-peptide antigens.

Improved efficacy of DNA vaccination against breast cancer by boosting with the repeat beta-hCG C-terminal peptide carried by mycobacterial heat-shock protein HSP65

Studies have demonstrated that active-specific immunotherapy has potential for controlling mammary tumor progression. Human chorionic gonadotropin (hCG) is expressed and extremely sensitive, easily detectable and highly correlated with breast cancer. We developed a gene vaccine using a plasmid vector to deliver the six copies of 10-amino acid residues of β-hCG 109–118 and β hCG C-terminal 37-amino acid (CTP37). BALB/c female mice were immunized with a combination of pCR-HBc-X6-βhCGCTP37 DNA vaccine and HSP-X6-βhCGCTP37 protein vaccine. pCR-HBc-X6-βhCGCTP37 DNA vaccine were injected intramuscularly three times, on days −46,−25 and −11 and HSP-X6-βhCGCTP37 protein were applied two times, 21 and 14 days before tumor cell challenge. We assessed a combined DNA and protein vaccine for its effect of against murine EMT6 mammary tumor cells. In this study, animals vaccinated DNA vaccination boosting with the repeat β-hCG C-terminal peptide carried by mycobacterial heat-shock protein HSP65 induced higher avidity antibodies and effectively inhibited the growth of tumor, compared with treatment using DNA alone or BCG priming HSP-X6-βhCGCTP37 protein boosting. The data presented demonstrate that improve immunogenicity of DNA vaccination by boosting with the repeat β-hCG C-terminal peptide carried by mycobacterial heat-shock protein HSP65, which should prove useful in the development of new DNA vaccine against growth factors for cancer immunotherapy.

Single dose of a vaccine based on DNA encoding mycobacterial hsp65 protein plus TDM-loaded PLGA microspheres protects mice against a virulent strain of Mycobacterium tuberculosis.

The high incidence of tuberculosis around the world and the inability of BCG to protect certain populations clearly indicate that an improved vaccine against tuberculosis is needed. A single antigen, the mycobacterial heat shock protein hsp65, is sufficient to protect BALB/c mice against challenge infection when administered as DNA vaccine in a three-dose-based schedule. In order to simplify the vaccination schedule, we coencapsulated hsp65-DNA and trehalose dimicolate (TDM) into biodegradable poly(DL-lactide-co-glycolide) (PLGA) microspheres. BALB/c mice immunized with a single dose of DNA-hsp65/TDM-loaded microspheres produced high levels of IgG2a subtype antibody and high amounts of IFN-gamma in the supernatant of spleen cell cultures. DNA-hsp65/TDM-loaded microspheres were also able to induce high IFN-gamma production in bulk lung cells from challenged mice and confer protection as effective as that attained after three doses of naked DNA administration. This new formulation also allowed a ten-fold reduction in the DNA dose when compared to naked DNA. Thus, this combination of DNA vaccine and adjuvants with immunomodulatory and carrier properties holds the potential for an improved vaccine against tuberculosis.

A link between Helicobacter pylori and/or Chlamydia spp. infections and atherosclerosis

Antibodies to Helicobacter pylori, Chlamydia spp. and Mycobacterium bovis were determined in patients with coronary heart disease, H. pylori-related dyspepsia, and tuberculosis, and healthy controls. Enzyme-linked immunosorbent assay was conducted with a glycine extract and CagA protein of H. pylori, chlamydial lipopolysaccharide and mycobacterial heat shock protein Hsp65. The prevalence of anti-glycine extract IgG in coronary heart disease patients was higher than in the tuberculosis group and controls, and the same as in dyspeptic patients. Anti-chlamydial IgG were more prevalent in the coronary heart disease group than in healthy subjects. There was no difference in the prevalence of anti-CagA IgG in the coronary heart disease group and controls or anti-Hsp65 IgG in the patients with coronary heart disease, dyspepsia, tuberculosis, and controls. Anti-glycine extract IgA (like anti-glycine extract IgG) were more prevalent in the coronary heart disease group than in the healthy group. The highest anti-glycine extract IgG/IgA and anti-chlamydial IgG titers were more frequent in coronary heart disease patients as compared with controls. Infections with H. pylori and Chlamydia spp. and enhanced production of antibodies to these pathogens may predispose to human atherosclerosis.

Helicobacter pylori infection in connective tissue disorders is associated with high levels of antibodies to mycobacterial hsp65 but not to human hsp60.

To investigate whether the Helicobacter pylori status influences levels of antibodies against mycobacterial heat shock protein (hsp) 65 and human hsp60 in systemic autoimmune diseases and to study the concentration of anti-H. pylori antibodies in autoimmune patients and healthy controls. Antibodies against human heat-shock protein hsp60, mycobacterial heat-shock protein hsp65 were analyzed by ELISA. Anti-Helicobacter antibodies were determined by enzyme immunoassay. There was a markedly higher prevalence of H. pylori infection in undifferentiated connective tissue disease (82%) (n = 33) and systemic sclerosis (78%) (n = 55) but not in systemic lupus erythematosus (n = 49), polymyositis/dermatomyositis (n = 14), rheumatoid arthritis (n = 21) or primary Raynaud's syndrome (n = 26) compared with controls (59%) (n = 349). In autoimmune diseases H. pylori infection was associated with elevated levels of antihsp65 (p =.008) but not of antihsp60. Anti-hsp65 levels were significantly higher in H. pylori-infected (n = 129) than in uninfected patients (n = 69) (p =.0007). These findings indicate that in autoimmune diseases the infection with the H. pylori bacterium is associated with increased concentration of antimycobacterial hsp65.