Generation of a Recombinant 65-Kilodalton Mannoprotein, a Major Antigen Target of Cell-Mediated Immune Response toCandida albicans

ABSTRACT A 65-kDa mannoprotein (CaMp65) has long been studied as a major, immunodominant antigen of the human opportunistic pathogenCandida albicans. An expression library of C. albicans was screened with serum from mice immunized with ScMp65 (ScW10), a Saccharomyces cerevisiae recombinant protein of about 48 kDa. This serum recognized the CaMp65 from a cell wall extract of C. albicans. After cloning and sequencing of the relevant C. albicans cDNA, an open reading frame encoding a protein of 379 amino acids was identified. Its deduced amino acid sequence showed regions of identity with all previously characterized tryptic fragments of CaMp65, as well as with the corresponding regions of ScMp65. A prepeptide of 32 amino acids with signal peptidase and Kex2 cleavage sites as well as a high number of potential O-glycosylation sites but no N-glycosylation sites or GPI anchor were observed in sequence studies of CaMp65. A putative adhesin RGD sequence was also present in the C-terminal region of the molecule. This triplet was absent in the ScMp65. The relevant gene (designatedCaMP65) was localized to chromosome R of C. albicans as determined by pulse-field gel electrophoresis. Northern blot analysis demonstrated that gene transcription was heat inducible and associated with germ-tube formation by the fungus. A recombinant, His6-tagged protein (rCaMp65) was expressed inEscherichia coli under an inducible promoter. After purification by nickel-chelate affinity chromatography, the recombinant product was detected as a 47-kDa protein band in immunoblots with the anti-ScMp65 serum, as well as with CaMp65-specific monoclonal antibodies. Both ScMp65 and CaMp65 were assayed for antigenic stimulation in cultures of peripheral blood mononuclear cells (PBMC) from 10 unselected human donors. While ScMp65 was substantially nonstimulatory, both rCaMp65 and the native CaMp65 were equally able to induce lymphoproliferation of the PBMC from all the donors. In addition, a number of CD4+ T-cell clones were generated using a C. albicans mannoprotein fraction as an antigenic stimulant. Several of these clones specifically responded to both the native and the recombinant C. albicans Mp65 but not to ScMp65. Thus, the recombinant Mp65 of C. albicans retains antigenicity and, as such, could be a valid, standardized reagent for serodiagnostic and immunological studies.

Download Full-text

Cloning, Expression, and Immunological Evaluation of Two Putative Secreted Serine Protease Antigens ofMycobacterium tuberculosis

Infection and Immunity ◽

10.1128/iai.67.8.3998-4007.1999 ◽

1999 ◽

Vol 67 (8) ◽

pp. 3998-4007 ◽

Cited By ~ 75

Author(s):

Yasir A. W. Skeiky ◽

Michael J. Lodes ◽

Jeffrey A. Guderian ◽

Raodoh Mohamath ◽

Teresa Bement ◽

...

Keyword(s):

Serine Protease ◽

Recombinant Proteins ◽

Mononuclear Cells ◽

Vaccine Development ◽

High Titer ◽

In Vitro Assays ◽

Protease Gene ◽

Expression Library ◽

Peripheral Blood Mononuclear ◽

Polyclonal Rabbit

ABSTRACT Culture filtrate proteins (CFP) of Mycobacterium tuberculosis have been shown to contain immunogenic components that elicit at least partial protective immunity againstMycobacterium infection. To clone genes encoding some of the immunogenic proteins, we made a high-titer rabbit anti-CFP serum and used it to screen an M. tuberculosis genomic expression library in Escherichia coli. In this paper, we describe the molecular cloning of two new protein components of CFP and identified them as members of the serine protease gene family. Their open reading frames contain N-terminal hydrophobic secretory signals consistent with their detection in CFP. The predicted molecular masses of the mature, fully processed forms of both antigens are ∼32 kDa, in agreement with their observed sizes on immunoblots of CFP probed with polyclonal rabbit antisera made to the recombinant proteins. Thus, these proteins have been designated MTB32A and MTB32B. Interestingly, and despite 66% amino acid sequence homology between the two proteins, polyclonal rabbit antisera made to each of the recombinant proteins were found to be specific for the respective immunizing antigens. The recombinant proteins were also evaluated in in vitro assays with donor peripheral blood mononuclear cells (PBMC) from healthy purified protein derivative (PPD)-positive individuals of diverse ethnic backgrounds. MTB32A but not MTB32B stimulated PBMC from healthy PPD-positive donors but not from PPD-negative donors to proliferate and secrete gamma interferon. MTB32A is encoded by a single-copy gene which is present in both virulent and avirulent strains of the M. tuberculosiscomplex and the BCG strain of Mycobacterium bovis but absent in the environmental mycobacterial species tested. In addition, nucleotide sequence comparison of mtb32a of the avirulent H37Ra strain and the virulent Erdman strain, as well as with the corresponding sequences (identified in the databases) of strain H37Rv and the clinical isolate CSU93, revealed 100% identity. MTB32A, therefore, represents a candidate for inclusion in subunit vaccine development. Finally, the possible role of MTB32 serine proteases as a virulence factor(s) during Mycobacterium spp. infection is discussed.

Download Full-text

Human and Murine Immune Responses to a NovelLeishmania major Recombinant Protein Encoded by Members of a Multicopy Gene Family

Infection and Immunity ◽

10.1128/iai.66.7.3279-3289.1998 ◽

1998 ◽

Vol 66 (7) ◽

pp. 3279-3289 ◽

Cited By ~ 112

Author(s):

John R. Webb ◽

Antonio Campos-Neto ◽

Pamela J. Ovendale ◽

Tricia I. Martin ◽

Erika J. Stromberg ◽

...

Keyword(s):

Immune Responses ◽

Leishmania Major ◽

Mononuclear Cells ◽

Blot Hybridization ◽

Interleukin 12 ◽

Predicted Amino Acid Sequence ◽

Cdna Expression Library ◽

Expression Library ◽

Peripheral Blood Mononuclear ◽

Amino Terminal

ABSTRACT Vaccination of BALB/c mice with Leishmania majorpromastigote culture filtrate proteins plus Corynebacterium parvum confers resistance to infection with L. major. To define immunogenic components of this protein mixture, we used sera from vaccinated mice to screen an L. major amastigote cDNA expression library. One of the immunoreactive clones thus obtained encoded a novel protein of L. major with a molecular mass of 22.1 kDa. The predicted amino acid sequence of this clone exhibited significant homology to eukaryotic thiol-specific-antioxidant (TSA) proteins. Therefore, we have designated this protein L. major TSA protein. Southern blot hybridization analyses indicate that there are multiple copies of the TSA gene in all species ofLeishmania analyzed. Northern blot analyses demonstrated that the TSA gene is constitutively expressed in L. majorpromastigotes and amastigotes. Recombinant TSA protein containing an amino-terminal six-histidine tag was expressed in Escherichia coli with the pET17b system and was purified to homogeneity by affinity chromatography. Immunization of BALB/c mice with recombinant TSA protein resulted in the development of strong cellular immune responses and conferred protective immune responses against infection with L. major when the protein was combined with interleukin 12. In addition, recombinant TSA protein elicited in vitro proliferative responses from peripheral blood mononuclear cells of human leishmaniasis patients and significant TSA protein-specific antibody titers were detected in sera of both cutaneous-leishmaniasis and visceral-leishmaniasis patients. Together, these data suggest that the TSA protein may be useful as a component of a subunit vaccine against leishmaniasis.

Download Full-text

Selection of Virus Variants and Emergence of Virus Escape Mutants after Immunization with an Epitope Vaccine

Journal of Virology ◽

10.1128/jvi.72.2.1403-1410.1998 ◽

1998 ◽

Vol 72 (2) ◽

pp. 1403-1410 ◽

Cited By ~ 68

Author(s):

Lorenzo Mortara ◽

Franck Letourneur ◽

Helene Gras-masse ◽

Alain Venet ◽

Jean-Gerard Guillet ◽

...

Keyword(s):

Amino Acids ◽

Mononuclear Cells ◽

Cytotoxic T Lymphocyte ◽

Target Cells ◽

Epitope Vaccine ◽

Peripheral Blood Mononuclear ◽

Gag Proteins ◽

Escape Mutants ◽

Selection Of ◽

Ctl Responses

ABSTRACT In this report, we assessed the evolution of the cytotoxic T-lymphocyte (CTL) response induced by an epitope vaccine. In two macaques immunized with a mixture of lipopeptides derived from simian immunodeficiency virus (SIV) Nef and Gag proteins, CTL responses were directed against the same, single epitope of the Nef protein (amino acids 128 to 137) presenting an alanine at position 136 (Nef 128-137/136A). However, after 5 months of SIV infection, peripheral blood mononuclear cells from both macaques lost their ability to be stimulated by autologous SIV-infected cells while still retaining their capacity to generate cytotoxic responses after specific Nef 128-137/136A peptide stimulation. The sequences of the pathogenic viral isolate used for the challenge showed a mixture of several variants. Within the Nef epitopic sequence from amino acids 128 to 137, 82% of viral variants expressed the epitopic peptide Nef 128-137/136A; the remaining variants presented a threonine at position 136 (Nef 128-137/136T). In contrast, sequence analysis of cloned proviral DNA obtained from both macaques 5 months after SIV challenge showed a different pattern of quasi-species variants; 100% of clones presented a threonine at position 136 (Nef 128-137/136T), suggesting the disappearance of viral variants with an alanine at this position under antiviral pressure exerted by Nef 128-137/136A-specific CTLs. In addition, 12 months after SIV challenge, six of eight clones from one macaque presented a glutamic acid at position 131 (Nef 128-137/131E+136T), which was not found in the infecting isolate. Furthermore, CTLs generated very early after SIV challenge were able to lyse cells sensitized with the Nef 128-137/136A epitope. In contrast, lysis was significantly less effective or even did not occur when either the selected peptide Nef 128-137/136T or the escape variant peptide Nef 128-137/131E+136T was used in a target cell sensitization assay. Dose analysis of peptides used to sensitize target cells as well as a major histocompatibility complex (MHC)-peptide stability assay suggested that the selected peptide Nef 128-137/136T has an altered capacity to bind to the MHC. These data suggest that CTL pressure leads to the selection of viral variants and to the emergence of escape mutants and supports the fact that immunization should elicit broad CTL responses.

Download Full-text

Molecular and Immunological Characterization ofMycobacterium tuberculosis CFP-10, an Immunodiagnostic Antigen Missing in Mycobacterium bovis BCG

Journal of Clinical Microbiology ◽

10.1128/jcm.38.9.3285-3290.2000 ◽

2000 ◽

Vol 38 (9) ◽

pp. 3285-3290 ◽

Cited By ~ 83

Author(s):

Davin C. Dillon ◽

Mark R. Alderson ◽

Craig H. Day ◽

Teresa Bement ◽

Antonio Campos-Neto ◽

...

Keyword(s):

T Cell ◽

Mononuclear Cells ◽

Immunoblot Analysis ◽

Expression Library ◽

Peripheral Blood Mononuclear ◽

Genomic Expression ◽

Purified Protein Derivative ◽

Acid Protein ◽

Smear Negative ◽

Amino Acid Protein

In order to identify antigens that may be used in the serodiagnosis of active tuberculosis (TB), we screened a Mycobacterium tuberculosis genomic expression library with a pool of sera from patients diagnosed with active pulmonary TB. The sera used lacked reactivity with a recombinant form of the M. tuberculosis38-kDa antigen (r38kDa), and the goal was to identify antigens that might complement r38kDa in a serodiagnostic assay. Utilizing this strategy, we identified a gene, previously designated lhp, which encodes a 100-amino-acid protein referred to as culture filtrate protein 10 (CFP-10). The lhp gene is located directly upstream of esat-6, within a region missing in M. bovis BCG. Immunoblot analysis demonstrated that CFP-10 is present in M. tuberculosis CFP, indicating that it is likely a secreted or shed antigen. Purified recombinant CFP-10 (rCFP-10) was shown to be capable of detecting specific antibody in a percentage of TB patients that lack reactivity with r38kDa, most notably in smear-negative cases, where sensitivity was increased from 21% for r38kDa alone to 40% with the inclusion of rCFP-10. In smear-positive patient sera, sensitivity was increased from 49% for r38kDa alone to 58% with the inclusion of rCFP-10. In addition, rCFP-10 was shown to be a potent T-cell antigen, eliciting proliferative responses and gamma interferon production from peripheral blood mononuclear cells in 70% of purified protein derivative-positive individuals without evident disease. The responses to this antigen argue for the inclusion of rCFP-10 in a polyvalent serodiagnostic test for detection of active TB infection. rCFP-10 could also contribute to the development of a recombinant T-cell diagnostic test capable of detecting exposure to M. tuberculosis.

Download Full-text

Importance of the Alanine Methyl Ester Side Chain for the Biological Activity Profile of Dual-Function Phenyl Phosphate Derivatives of Bromo-Methoxy-Zidovudine

Antiviral Chemistry and Chemotherapy ◽

10.1177/095632020001100103 ◽

2000 ◽

Vol 11 (1) ◽

pp. 31-39 ◽

Cited By ~ 5

Author(s):

TK Venkatachalam ◽

OJ D'Cruz ◽

FM Uckun

Keyword(s):

Amino Acids ◽

Mononuclear Cells ◽

Human Peripheral Blood ◽

Side Chain ◽

Computer Assisted ◽

Dual Function ◽

Peripheral Blood Mononuclear ◽

Derivatives Of ◽

Anti Hiv ◽

Phenyl Moiety

In a systematic search for developing a virucidal spermicide with potent anti-human immunodeficiency virus (HIV) and spermicidal activities, we synthesized and evaluated 14 phosphoramidate derivatives of 5-bromo-6-methoxy-zidovu-dine (PP-BMZ) with differing amino acid ester side chains and para substitutions on the phenyl moiety. Anti-HIV activity was tested by measuring viral p24 antigen production as a marker of viral replication in HIV-1-infected human peripheral blood mononuclear cells. The effect of various PP-BMZ compounds on human sperm motion kinematics was analysed by computer-assisted sperm analysis. Varying the Ala side chain of the phosphoramidate group to other non-polar amino acids, including the cyclic amino acids proline and tryptophan, led to significant alterations in both anti-HIV and spermicidal activities. Our findings highlight the necessity of the Ala side chain and the presence of an electron-withdrawing para-bromo substi-tutent on the phenyl moiety in addition to the bromo-methoxy functional groups on the thymine ring for the PP-BMZ compounds to be effective virucidal spermicides. These membrane permeable dual-function nucleoside analogues may provide the basis for a new strategy aimed at prevention of the sexual transmission of HIV while providing fertility control for women.

Download Full-text

Identification of Three Novel Superantigen-Encoding Genes in Streptococcus equi subsp. zooepidemicus, szeF, szeN, and szeP

Infection and Immunity ◽

10.1128/iai.00751-10 ◽

2010 ◽

Vol 78 (11) ◽

pp. 4817-4827 ◽

Cited By ~ 42

Author(s):

Romain Paillot ◽

Alistair C. Darby ◽

Carl Robinson ◽

Nicola L. Wright ◽

Karen F. Steward ◽

...

Keyword(s):

Horizontal Transfer ◽

Mononuclear Cells ◽

Opportunistic Pathogen ◽

Mitogenic Activity ◽

Peripheral Blood Mononuclear ◽

Necrosis Factor Alpha ◽

Streptococcus Equi ◽

Factor Alpha ◽

Encoding Genes

ABSTRACT The acquisition of superantigen-encoding genes by Streptococcus pyogenes has been associated with increased morbidity and mortality in humans, and the gain of four superantigens by Streptococcus equi is linked to the evolution of this host-restricted pathogen from an ancestral strain of the opportunistic pathogen Streptococcus equi subsp. zooepidemicus. A recent study determined that the culture supernatants of several S. equi subsp. zooepidemicus strains possessed mitogenic activity but lacked known superantigen-encoding genes. Here, we report the identification and activities of three novel superantigen-encoding genes. The products of szeF, szeN, and szeP share 59%, 49%, and 34% amino acid sequence identity with SPEH, SPEM, and SPEL, respectively. Recombinant SzeF, SzeN, and SzeP stimulated the proliferation of equine peripheral blood mononuclear cells, and tumor necrosis factor alpha (TNF-α) and gamma interferon (IFN-γ) production, in vitro. Although none of these superantigen genes were encoded within functional prophage elements, szeN and szeP were located next to a prophage remnant, suggesting that they were acquired by horizontal transfer. Eighty-one of 165 diverse S. equi subsp. zooepidemicus strains screened, including 7 out of 15 isolates from cases of disease in humans, contained at least one of these new superantigen-encoding genes. The presence of szeN or szeP, but not szeF, was significantly associated with mitogenic activity in the S. equi subsp. zooepidemicus population (P < 0.000001, P < 0.000001, and P = 0.104, respectively). We conclude that horizontal transfer of these novel superantigens from and within the diverse S. equi subsp. zooepidemicus population is likely to have implications for veterinary and human disease.

Download Full-text