An HLA-directed molecular and bioinformatics approach identifies new HLA-A11 HIV-1 subtype E cytotoxic T lymphocyte epitopes in HIV-1-infected Thais.

Only limited cytotoxic T lymphocyte (CTL) epitope mapping has been done in nonsubtype B HIV-infected persons. We used molecular immunogenetic tools to determine HIV-specific CTL responses in HIV-1 Env subtype E-infected female sex workers (FSWs) from northern Thailand, where more than 50% of the population is HLA-A11 positive. EpiMatrix, a computer-based T cell epitope prediction algorithm, and a manual editing approach were used to predict 77 possible HLA-A11 CTL epitopes in HIV-1, some of which were conserved between subtypes B and E. MHC binding of these peptides was determined in an HLA-A11 stabilization assay, and binding peptides were tested for CTL recognition in eight HLA-A11-positive FSWs. Subtype E versions of known HLA-A2 subtype B HIV epitopes were also tested in four HLA-A2 positive FSWs. CTL responses were detected in all HLA-A11-positive and in three of four HLA-A2-positive persons. Among the 12 FSWs responses to peptides were found to Pol in 9 (75%), Env in 7 (58%), Nef in 5 (42%), and Gag in 5 (42%), and to conserved epitopes in 8 (67%). To identify HLA-A11 CTL epitopes in the absence of prediction tools, it would have been necessary to test almost 3000 10-mer peptides. EpiMatrix and manual predictions reduced this number to 77, of which 26 were MHC binding and 12 were CTL epitopes. Six of these HLA-A11 CTL epitopes have not been previously reported and are located in RT, gp120, and gp41. This report of CTL responses in subtype E-infected individuals defines epitopes that may be useful in HIV pathogenesis or vaccine studies.

HIV-specific cytotoxic T lymphocytes, HLA-A11, and chemokine-related factors may act synergistically to determine HIV resistance in CCR5 delta32-negative female sex workers in Chiang Rai, northern Thailand.

Understanding how highly HIV-exposed individuals remain HIV uninfected may be useful for HIV vaccine design and development of new HIV prevention strategies. To elucidate mechanisms associated with resistance to HIV infection, immunologic and genetic factors were examined in 14 HIV-exposed but persistently seronegative (HEPS) female sex workers from Chiang Rai, northern Thailand and in ethnically matched, HIV-positive (n = 9) and HIV-negative women (n = 9). The HEPS women were identified in a study of commercial sex workers who had an HIV-1 incidence of 20.3 per 100 person-years. A high frequency of HLA-A11 was observed in HEPS women (86%) compared with northern Thai controls (56%). HIV-specific cytotoxic T lymphocyte (CTL) lytic responses were detected in cryopreserved peripheral blood mononuclear cells (PBMCs), using HLA-A-matched subtype E HIV-1 peptides in four of seven (57%) HEPS women, eight of eight HIV-positive women, and zero of nine HIV-negative unexposed controls (p = 0.019 HEPS women vs. HIV-negative controls). CTL lysis levels were low, but responses were detected to peptides from Nef, Pol, Gag, and Env. Nef responses predominated in HEPS women. Compared with controls, HEPS women tended to have higher frequencies of CCR5 promotor 59402GG and SDF-1 3'UTR 801A genotypes known to influence HIV transmission or course of disease. HEPS women also had higher levels of spontaneous RANTES production by PBMCs than other groups. Each of these factors could potentially contribute to HIV resistance. As most HEPS women had one or more of these factors, they may prevent HIV infection synergistically by blocking HIV cell entry, delaying its dissemination, or killing HIV-infected cells.

In vitro effect of Derris scandens on normal lymphocyte proliferation and its activities on natural killer cells in normals and HIV-1 infected patients.

We investigated the effect of Derris scandens hydroalcoholic extract on lymphocyte proliferation, natural killer (NK) cell activity and secretion of IL-2 and IL-4. Lymphocyte proliferative response of normal individuals was significantly increased at concentrations of 10 ng/ml, 100 ng/ml, 1 microg/ml and 5 microg/ml, whereas the response was significantly decreased at 100 microg/ml. D. scandens at the concentrations of 10 ng/ml, 100 ng/ml, 1 microg/ml and 10 microg/ml significantly enhanced the function of NK cells of normal individuals. The NK cell activity of HIV-infected individuals was significantly increased at a concentration of 10 microg/ml. Furthermore, the extract was shown to induce the IL-2 secretion from normal peripheral blood mononuclear cells (PBMC), whereas the IL-4 was not induced in the presence of the D. scandens extract. Our data suggested that the hydroalcoholic extract of D. scandens possesses in vitro immunostimulating activity on human immunocompetent and immunocompromised PBMC.

Single-step purification of Proteus mirabilis urease accessory protein UreE, a protein with a naturally occurring histidine tail, by nickel chelate affinity chromatography.

Proteus mirabilis urease, a nickel metalloenzyme, is essential for the virulence of this species in the urinary tract. Escherichia coli containing cloned structural genes ureA, ureB, and ureC and accessory genes ureD, ureE, ureF, and ureG displays urease activity when cultured in M9 minimal medium. To study the involvement of one of these accessory genes in the synthesis of active urease, deletion mutations were constructed. Cultures of a ureE deletion mutant did not produce an active urease in minimal medium. Urease activity, however, was partially restored by the addition of 5 microM NiCl2 to the medium. The predicted amino acid sequence of UreE, which concludes with seven histidine residues among the last eight C-terminal residues (His-His-His-His-Asp-His-His-His), suggested that UreE may act as a Ni2+ chelator for the urease operon. To exploit this potential metal-binding motif, we attempted to purify UreE from cytoplasmic extracts of E. coli containing cloned urease genes. Soluble protein was loaded onto a nickel-nitrilotriacetic acid column, a metal chelate resin with high affinity for polyhistidine tails, and bound protein was eluted with a 0 to 0.5 M imidazole gradient. A single polypeptide of 20-kDa apparent molecular size, as shown by sodium dodecyl sulfate-10 to 20% polyacrylamide gel electrophoresis, was eluted between 0.25 and 0.4 M imidazole. The N-terminal 10 amino acids of the eluted polypeptide exactly matched the deduced amino acid sequence of P. mirabilis UreE. The molecular size of the native protein was estimated on a Superdex 75 column to be 36 kDa, suggesting that the protein is a dimer. These data suggest that UreE is a Ni(2)+-binding protein that is necessary for synthesis of a catalytically active urease at low Ni(2+) concentrations.

Sequence of the Proteus mirabilis urease accessory gene ureG.

We report the sequence of ureG, an accessory gene that is a part of the ure gene cluster of uropathogenic Proteus mirabilis and required for full enzymatic activity of urease. The 615-bp open reading frame predicts a M(r) 22,374 polypeptide, which contains a consensus amino acid (aa) sequence for ATP-binding. The polypeptide shares sequence homology with UreG of Escherichia coli (93% of identical aa), Klebsiella aerogenes (59%) and Helicobacter pylori (59%).

Proteus mirabilis urease: histidine 320 of UreC is essential for urea hydrolysis and nickel ion binding within the native enzyme.

Proteus mirabilis urease, a nickel-containing enzyme, has been established as a critical virulence determinant in urinary tract infection. An amino acid sequence (residues 308 to 327: TVDEHLDMLMVCHHLDPSIP) within the large urease subunit, UreC, is highly conserved for every urease examined thus far and has been suggested to reside within the enzyme active site. Histidine residues have been postulated to play a role in catalysis by coordinating Ni2+ ions. To test this hypothesis, oligonucleotide-directed mutagenesis was used to change amino acid His-320 to Leu-320 within UreC. The base change (CAT for His-320 to CTT for Leu-320) was confirmed by DNA sequencing. The recombinant and mutant proteins were expressed at similar levels in Escherichia coli as detected by Western blotting (immunoblotting) of denaturing and nondenaturing gels. Specific activities of the enzymes were quantitated after partial purification. Strains expressing the mutant enzyme showed no detectable activity, whereas strains expressing the recombinant enzyme hydrolyzed urea at 149 mumol of NH3 per min per mg of protein. In addition, the mutant enzyme was able to incorporate only about one-half (58%) of the amount of 63Ni2+ incorporated by the active recombinant enzyme. While the mutation of His-320 to Leu-320 within UreC does not affect expression or assembly of urease polypeptide subunits UreA, UreB, and UreC His-320 of UreC is required for urea hydrolysis and proper incorporation of Ni2+ into apoenzyme.

Lymphocyte subsets in human encephalitic and paralytic rabies.

Lymphocyte subsets of 7 patients with encephalitic and paralytic rabies were determined by immunocytochemical techniques using mouse monoclonal antibodies. Almost all patients had diminished mononuclear cells of Leu 7 phenotype (natural killer cells). Cells of Leu 12 marker (B cells) were decreased in 3 paralytic rabies patients compared with those of 4 patients in the encephalitic group.

Comparative immunogenicity study of four plasma-derived hepatitis B vaccines in Thai young adults.

Immunogenicity of four plasma-derived hepatitis B vaccines (Merck, Sharp and Dohme, Pasteur, Dutch CLB and Korean Cheil-Sugar) was compared in Thai young adults. After primoimmunization, only the Merck and Pasteur vaccines could achieve greater than 90% seroconversion (i.e. anti-HBs greater than or equal to 10 mIU ml-1) whereas both the CLB and Korean vaccines needed a fourth dose to achieve this level of seroconversion. The anti-HBs titres of both heat-inactivated vaccines (CLB and Korean) were also significantly lower than those of the other two vaccines. We propose that the HBsAg content in both heat-inactivated vaccines should be increased and a booster (fourth) dose should be given in order to enhance their immunogenicities.

Immunoenhancement with combined rabies and aluminium-adjuvanted tetanus vaccines.

The effect of combining tetanus toxoid in the same syringe with purified Vero cell rabies vaccine (PVRV) was investigated in the 2-1-1 regimen of PVRV. The 2-1-1 regimen alone was as immunogenic as the five-dose regimen, while saving one dose of vaccine and two clinic visits. When aluminium hydroxide-adsorbed tetanus toxoid was used to dissolve PVRV on days 0 (one of the two doses) and 21, the anti-rabies antibody was significantly increased. Aluminium-free tetanus toxoid was ineffective, suggesting that immunoenhancement was due to aluminium adjuvant. In addition, anti-tetanus antibody was unaffected and the side-effects were not increased by such mixing.

Immunologic study of human encephalitic and paralytic rabies. Preliminary report of 16 patients.

Lymphocyte proliferation tests to rabies antigen and myelin basic protein were performed on peripheral blood lymphocytes from nine patients with the encephalitic form and on seven with the paralytic form of human rabies. Six of the nine patients with encephalitis had proliferative responses to rabies antigen, whereas all of the patients with paralysis had no response. Two patients in each group also had a proliferative response to myelin basic protein. The myelin basic protein-reactive patients had a more rapidly fatal disease than the non-reactive patients. This preliminary study suggests that host immune responses may influence the clinical manifestations and course in human rabies.

Effect of inosiplex on the humoral and cell-mediated immune responses to intradermal human diploid cell rabies vaccine.

Antigen-stimulated lymphocyte transformation was studied in recipients of intradermal human diploid cell rabies vaccine (HDCV). HDCV was administered intradermally at 8 different anatomical sites, 0.1 ml each, on day 0; followed by another 4-site injection on day 7. Rabies antigen-stimulated in vitro proliferative response was evident as early as 7 days after starting immunization. It reached a peak on day 14 and had declined by day 28. The cellular proliferative response preceded and roughly correlated with the antirabies antibody response. Simultaneous administration of inosiplex, an antiviral and immunopotentiating drug, during the first 10 days of intradermal HDCV immunization did not result in heightened antibody titres or cell-mediated immune response to the vaccine. The number of T cells and the lymphocyte proliferative response to phytohaemagglutinin in inosiplex-treated vaccinees were similarly not significantly different from untreated controls. Our results confirm other previous findings that a specific cell-mediated immune response can be consistently and rapidly induced by an intradermal regimen of HDCV immunization. The addition of inosiplex to this regimen did not enhance the humoral or cell-mediated immune responses to the vaccine. The apparent lack of immunostimulating effect of inosiplex in this setting may be the result of several factors such as the immunization schedule and the immunologic parameters examined.

Cell-mediated immune response following intracutaneous immunisation with human diploid cell rabies vaccine.

Specific cell-mediated immune response (CMIR) against rabies antigens was studied in recipients of two regimens of human diploid cell rabies vaccine (HDCV) using the antigen-stimulated lymphocyte transformation test (LTT) as a measure of CMIR. Reconstituted HDCV could be conveniently used as the in vitro stimulating antigen and the response was antigen-dependent. Conventional intramuscular immunization with full-dose HDCV resulted in positive LTT as early as 14 days after starting immunisation, and peaked on day 28. Intracutaneous immunisation with 0.1 ml of HDCV at four sites on days 0, 3 and 7 was a more efficient means of inducing specific lymphocyte response. Specific CMIR was evident as early as seven days and became maximal on day 14. In addition to the more rapid induction of specific CMIR, our intracutaneous regimen also resulted in a brisker and higher antibody response than the intramuscular regimen. The peak antibody level of the intracutaneous regimen was reached on day 14 whereas that of the intramuscular regimen was reached on day 28 and the geometric mean antibody titre on day 14 of the intracutaneous route was significantly higher than that of the intramuscular regimen. We therefore conclude that our closely spaced intracutaneous immunisation with HDCV was effective both in the induction of specific antibodies and the cell-mediated immune response.