Philip S. Shepherd

Proliferative T cell responses to the human papillomavirus type 16 E7 protein in women with cervical dysplasia and cervical carcinoma and in healthy individuals

The levels of proliferative T cell responses to peptides representing the human papillomavirus type 16 (HPV-16) E7 protein have been measured using short-term T cell lines derived from peripheral blood of healthy women and those with cervical dysplasias and carcinoma of the cervix. In healthy individuals 47 percent (7/15) responded predominantly to the N- and C-terminal regions of the protein and 6/7 responders were to a single peptide between amino acids 80-94. In comparison 29 percent (9/31) of women with cervical dysplasia responded to HPV-16 E7, with a significantly reduced response to both the N- and C-terminal regions (P = 0.03 and 0.038, respectively). A higher proportion of responders was found in patients with high grade lesions (56 percent, 5/9) versus those with atypical or low grade histology (20 percent, 4/20) and the response to a single peptide between amino acids 75-94 was also increased in this patient group (P = 0.044). This may be a reflection of higher levels of current or previous exposure to HPV-16 in patients with high grade lesions. Correlation of T cell responses with HPV DNA type (detected by PCR of cervical biopsy tissue) showed that 3/9 (33 percent) HPV-16 DNA-positive individuals responded. This suggests that E7 may not be the dominant target of the immune response or that the response to E7 is down-regulated in these patients. In addition 4/18 (22 percent) HPV-16 DNA-negative individuals responded, suggesting that their T cells may have been primed by previous exposure to HPV-16 or that a cross-reactive response was detected. Proliferative T cell responses to both HPV-16 E7 and L1 were reduced in women with cervical carcinoma in comparison to those with cervical dysplasia and healthy controls. The observed down-regulation of responses to HPV-16 E7 in women with cervical dysplasia and cervical carcinoma may reflect an altered functional balance between subsets of T helper cells in HPV-16 infections.

MONOCLONAL ANTIBODIES THAT RECOGNIZE THE NATIVE HUMAN THYROTROPIN RECEPTOR

Monoclonal antibodies have been produced that recognize the native human thyrotropin receptor by using a sensitive screening protocol based on flow cytofluorimetry combined with recombinant eukaryotic cells expressing high levels of the full-length functional receptor. The more standard screening method of ELISA preferentially selected antibodies that only reacted with the denatured receptor. Mice were immunized with recombinant receptor produced in either eukaryotic or prokaryotic systems; after screening and cloning, three stable hybridoma lines were established. An IgM antibody (7B5) produced in response to the eukaryotic material recognized only the native receptor (by flow cytofluorimetry) and did not react with denatured material on ELISA or immunoblotting, suggesting that its epitope is conformational. In contrast, two IgG1 antibodies (2C11 and 3B12) produced in response to the prokaryotic material recognized both native and denatured receptor (by flow cytofluorimetry, immunoprecipitation and immunoblotting). The use of different recombinant constructs in the immunoblotting procedure allowed the epitopes for both the IgG1 antibodies to be assigned to the region 125-369. None of the antibodies stimulated production of cAMP by recombinant cells expressing the full-length functional receptor, but one of the IgG1 antibodies (2C11) did inhibit binding of radiolabelled thyrotropin to these same cells. These antibodies, and others that can now be produced with this screening protocol, will help define the relationship between structure and function of this important receptor.

Identification of immunogenic regions of the major coat protein of human papillomavirus type 16 that contain type-restricted epitopes.

We have identified regions of the major capsid protein, L1, of the human papillomavirus (HPV) type 16 (HPV-16 L1), that are recognized by five monoclonal antibodies (MAbs) raised to a bacterial fusion protein containing residues 172 to 375 of HPV-16 L1. All five MAbs recognized HPV-16-infected tissue sections by immunohistochemistry, but not sections infected with HPV-1a (cutaneous warts), HPV-6b or -11 (genital warts). MAbs 3D1, 5A4 and 1D6 also recognized HPV-2-infected sections (cutaneous warts); MAb 8C4 recognized only sections containing HPV-16. Four MAbs (8C4, 3D1, 1D6 and 5A4) recognized a synthetic peptide corresponding to residues 269 to 284 of HPV-16 L1; within this region a minimum antibody binding site was identified, a tripeptide 276 to 278. However the complete epitope appears to extend beyond these residues and beyond HPV-16 L1 (269 to 284). The fifth MAb, 1C6, recognized bacterial fusion proteins containing HPV-6b L1, -16 L1 or -18 L1 using immunoblots, yet appeared HPV-16-specific when tested on infected tissue sections. This MAb recognized five amino acids within a different region of HPV-16 L1 (residues 299 to 313).

Antibody-Targeted Lethal Photosensitization of Porphyromonas gingivalis

ABSTRACT We have previously demonstrated that Porphyromonas gingivalis is susceptible to killing by toluidine blue O (TBO) when irradiated with light from a helium-neon (HeNe) laser. The aim of this study was to determine whether a TBO-antibody conjugate (Ab-TBO) could be used to specifically target P. gingivalis to lethal photosensitization in the presence ofStreptococcus sanguis or human gingival fibroblasts (HGFs). When a mixture of P. gingivalis and S. sanguiswas exposed to 4 μg of TBO/ml and irradiated with HeNe laser light, there were 1.5- and 4.0-log10-unit reductions in the viable counts, respectively. In contrast, when TBO was conjugated with a murine monoclonal antibody against P. gingivalislipopolysaccharide, the reductions in viable counts of P. gingivalis and S. sanguis amounted to 5.0 and 0.1 log10 units, respectively. Lethal photosensitization ofP. gingivalis in the presence of HGFs using unconjugated TBO resulted in a 0.7-log10-unit reduction in P. gingivalis viable counts and a 99% reduction in the incorporation of tritiated thymidine ([3H]Tdr) by the HGFs. In contrast, when the Ab-TBO conjugate was used, there was a 100% reduction in P. gingivalis viable counts but no significant reduction in the incorporation of [3H]Tdr by HGFs. These results demonstrate that specific targeting of P. gingivalis can be achieved using TBO conjugated to a monoclonal antibody raised against a cell surface component of this organism.

Nuclear Targeting of Porphyromonas gingivalis W50 Protease in Epithelial Cells

ABSTRACT Porphyromonas gingivalis is an important pathogen associated with destructive periodontal disease and is able to invade the epithelial cell barrier. Its cysteine proteases are recognized as major virulence factors, and in this study, we examined the interaction of the arginine-specific protease with epithelial cells in culture. Three cell lines (KB, HeLa, and SCC4) were incubated with strain W50 culture supernatant; stained with monoclonal antibody 1A1, which recognizes an epitope on the adhesin (β) component of the cysteine protease-adhesin (α/β) heterodimer; and viewed using immunofluorescence microscopy. Within 1 h, the protease traversed the plasma membrane and was localized around the nucleus before becoming concentrated in the cytoplasm after 24 to 48 h. In contrast, the purified arginine-specific heterodimeric protease (HRgpA) rapidly entered the nucleus within 15 to 30 min. This nuclear targeting (i) was seen with active and Nα-p-tosyl-l-lysine chloromethyl ketone (TLCK)-inactivated HRgpA, indicating it was independent of the proteolytic activity; (ii) occurred at both 4 and 37°C; and (iii) failed to occur with the monomeric protease (RgpAcat), indicating the importance of the adhesin chain of the HRgpA protease to this process. Rapid cell entry was also observed with recombinant catalytic (α) and adhesin (β) chains, with the latter again targeting the nuclear area. After 48 h of incubation with HRgpA, significant dose-dependent stimulation of metabolic activity was observed (measured by reduction of 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide), and a doubling of mitotic activity combined with the presence of apoptotic cells indicated that HRgpA may interfere with cell cycle control mechanisms. These effects were seen with both active and TLCK-inactivated protease, confirming that they were not dependent on proteolytic activity, and thus provide new insights into the functioning of this P. gingivalis protease.

Reactivities of polyclonal and monoclonal antibodies raised to the major capsid protein of human papillomavirus type 16.

Polyclonal and monoclonal antibodies have been raised against a fusion protein containing beta-galactosidase and part of the major capsid protein L1 of the human papillomavirus (HPV) type 16. The polyclonal antibodies cross-reacted with the L1 protein of several HPV types including HPV-1, -2, -6 and -11 when reacted with virus-infected tissue sections, and with HPV-6 and -18 L1 fusion proteins on Western blotting. Monoclonal antibodies against the L1 fusion protein of HPV-16 reacted only with HPV-16 L1 fusion proteins on Western blots and with HPV-16-containing biopsy sections as assessed by in situ DNA-DNA hybridization. These antibodies did not detect HPV-6 L1 protein after Western blotting or in HPV-6-infected tissue sections, although one did react with an HPV-18 fusion protein after Western blotting. The monoclonal antibodies were able to detect HPV-16 antigens in routine formaldehyde-fixed, wax-embedded sections of cervical intraepithelial neoplasia sections. HPV-16 L1 proteins were seen in one-third of biopsies that were positive using the polyclonal cross-reacting antisera. Polyclonal antibodies to fusion proteins containing part of the minor capsid protein L2 of HPV-6 or -16 appeared to be more type-specific as no cross-reactivity was seen when these antibodies were reacted with HPV-1- and -2-infected tissue sections.

T cell responses to the human papillomavirus type 16 E7 protein in mice of different haplotypes

The response of murine T cells to the E7 molecule of human papillomavirus type 16 (HPV-16) was studied using eight different mouse strains of six distinct H-2 haplotypes. HPV-16 E7 protein was prepared as a fusion protein with glutathione-S-transferase, purified by affinity chromatography and used for immunization. Cells from the lymph nodes were cultured with whole fusion protein, glutathione-S-transferase or HPV-16 E7 protein synthetic peptides. All the mouse strains tested, with the exception of BALB/c, recognized the E7 molecule, as evidenced by a proliferative response to at least two of the peptides. The profile of responses to peptides varied between and within a strain, but five distinct immunodominant regions could be identified. These regions were defined on the basis of a reaction to one or more peptides in a given part of the E7 molecule by at least four strains. The five regions were encompassed by amino acid residues 1 to 9, 17 to 32, 42 to 59, 62 to 77 and 87 to 98. The findings suggest that in an outbred population, such as man, the E7 molecule of HPV-16 would be recognized by a large proportion of the population. However, the poor response of two mouse strains [B10.RIII (71NS) and BALB/c] could also have a corollary in man.

A functional site on the human TSH receptor: a potential therapeutic target in Graves’ disease

objective  Identifying sites on the TSH‐receptor that are involved in the pathological stimulation of the thyroid by autoantibodies in Graves’ disease would aid the development of new therapies. We tested a series of monoclonal antibodies that recognize the native receptor for their ability to inhibit stimulation of the receptor in vitro.

Human papillomavirus antigens and T-cell recognition

This review will summarize what is known about natural T-cell responses to human papillomavirus infections, including potential mechanisms for their dysregulation which may lead to the development of disease. We will also describe new strategies to enhance human papillomavirus specific T-cell responses following vaccination that are currently in development and recent reports on human vaccine trials with particular regard to the generation of appropriate T-cell responses.

An Immunodominant Region in HPV16.L1 Identified by T Cell Responses in Patients with Cervical Dysplasias

Human papillomaviruses are truly epitheliotropic and give rise to skin and mucosal. lesions. How the host controls and eliminates HPV infections at these surfaces remains largely unknown. In the genital. tract HPV has been shown to cause genital. warts, cervical. dysplasias and carcinomas of the cervix. Most cases of genital. warts and early lesions of cervical. dysplasias will spontaneously regress if left untreated suggesting that the host mounts a protective response against the virus. High grade dysplasias and tumours appear not to be controlled despite the fact that high risk HPV types (16,18,31, 33 etc.) are found in both situations. The role of immune mechanisms in HPV infections can now be examined where previously it was impossible through the development of new assay techniques. This has come about following the availability of good antigen sources, namely HPV capsids for B cell (serology) studies and recombinant HPV proteins and synthetic peptides for T cell work. Our present study looks for proliferative T cell responses to HPV16.L1 in patients with cervical. dysplasias by generating short term T cell lines from their peripheral. blood in vitro and using these to map immunodominant T cell epitopes on the molecule with overlapping synthetic peptides.