Intradermal delivery of vaccines: potential benefits and current challenges.

Delivery of vaccine antigens to the dermis and/or epidermis of human skin (i.e. intradermal delivery) might be more efficient than injection into the muscle or subcutaneous tissue, thereby reducing the volumes of antigen. This is known as dose-sparing and has been demonstrated in clinical trials with some, but not all, vaccines. Dose-sparing could be beneficial to immunization programmes by potentially reducing the costs of purchase, distribution and storage of vaccines; increasing vaccine availability and effectiveness. The data obtained with intradermal delivery of some vaccines are encouraging and warrant further study and development; however significant gaps in knowledge and operational challenges such as reformulation, optimizing vaccine presentation and development of novel devices to aid intradermal vaccine delivery need to be addressed. Modelling of the costs and potential savings resulting from intradermal delivery should be done to provide realistic expectations of the potential benefits and to support cases for investment. Implementation and uptake of intradermal vaccine delivery requires further research and development, which depends upon collaboration between multiple stakeholders in the field of vaccination.

Thermostable formulations of a hepatitis B vaccine and a meningitis A polysaccharide conjugate vaccine produced by a spray drying method.

Thermostable vaccines promise to simplify the logistics of vaccine distribution and expand the immunization coverage. In this study, a pilot-scale spray drying process was developed and used to produce glassy state formulations of a recombinant hepatitis B (HepB) vaccine containing aluminum adjuvant and Neisseria meningitidis A (MenA) protein-polysaccharide conjugate vaccine, representing two common types of subunit vaccines in use today: the spray-dried HepB vaccine formulations were stable for at least 24 months at 37 degrees C while several MenA vaccine formulations exhibited complete stability at temperatures up to 60 degrees C. This study demonstrates the feasibility of producing thermostable vaccines with advanced processing and formulation technologies.

Comparative safety and efficacy of two high dose regimens of oral paracetamol in healthy adults undergoing third molar surgery under local anaesthesia.

This study compared the efficacy and safety of single oral doses of 60 mg/kg and 90 mg/kg paracetamol in fit young adult patients undergoing third molar extractions. The study was a randomised, blinded, crossover design on 20 young, fit adults. Paracetamol was administered 30 minutes prior to the surgical extraction of the teeth, which was done under intravenous sedation and local anaesthesia. There were no clinically or statistically significant differences in the pain scores between 60 mg/kg or 90 mg/kg doses until the intake of rescue analgesics. There was a reduction in factor VII activity with 90 mg/kg dose compared to 60 mg/kg dose. It may be concluded that the 90 mg/kg dose, though safe, does not offer any advantages over 60 mg/kg dose of paracetamol in young fit adults undergoing third molar surgery.

Effect of TA-CIN (HPV 16 L2E6E7) booster immunisation in vulval intraepithelial neoplasia patients previously vaccinated with TA-HPV (vaccinia virus encoding HPV 16/18 E6E7).

Heterologous prime-boost vaccination schedules employing TA-HPV, a vaccinia virus encoding HPV 16/18 E6 and E7, in combination with TA-CIN, an HPV 16 L2E6E7 fusion protein, may offer advantages over the use of either agent alone for the immunotherapy of human papillomavirus (HPV) type 16-associated vulval intraepithelial neoplasia (VIN). In the present study, 10 women with HPV 16-positive high grade VIN, previously primed with TA-HPV, received three booster immunisations with TA-CIN. All but one demonstrated HPV 16-specific proliferative T-cell and/or serological responses following vaccination. Three patients additionally showed lesion shrinkage or symptom relief, but no direct correlation between clinical and immunological responses was seen.

Enhancement of human papillomavirus (HPV) type 16 E6 and E7-specific T-cell immunity in healthy volunteers through vaccination with TA-CIN, an HPV16 L2E7E6 fusion protein vaccine.

TA-CIN is a vaccine that comprises the human papillomavirus (HPV) type 16 L2, E6 and E7 as a single fusion protein. In a mouse model, TA-CIN effectively prevented outgrowth of HPV16-positive tumour cells. To assess the safety and immunogenicity of TA-CIN, a dose escalating (26, 128, 533 micro g), double blind and placebo-controlled phase I study was conducted in 40 healthy volunteers. TA-CIN was administered without adjuvant by intramuscular injection on weeks 0, 4 and 8. No serious adverse events of the vaccination were reported during the study. Both IgG antibodies and proliferative responses against TA-CIN were elicited at all three doses. More importantly, T-cell immunity against the HPV16 E6 and E7 oncoproteins was detected by IFN gamma ELISPOT in 8/11 evaluable subjects vaccinated with the 533 micro g dose.

The barriers to effective management of heart failure in general practice.

BACKGROUND: Several studies have shown that most patients with heart failure are not investigated and treated according to published guidelines. More effective management could reduce both mortality and morbidity from heart failure. AIM: To identify the reasons for gaps between recommended and actual management of heart failure in general practice. DESIGN OF STUDY: A nominal group technique was used to elicit general practitioners' (GPs') perceptions of the reasons for differences between observed and recommended practice. SETTING: Ten Medical Research Council General Practice Framework practices in the North Thames region. METHOD: Data were collected on the investigation and treatment of heart failure in the 10 participating practices and presented to 49 GPs and 10 practice nurses from those practices. RESULTS: Of the 674 patients requiring echocardiograms, 226 were referred for echocardiography (34%), and 183/391 (47%) with probable heart failure were prescribed angiotensin-converting enzyme inhibitors. A wide variety of barriers were elicited. The main barrier to the use of echocardiograms in the diagnosis of heart failure was lack of open access. The main barrier to the use of angiotensin-converting enzyme inhibitors in treating heart failure was GPs' concerns about their possible adverse effects. CONCLUSION: The barriers to the effective management of heart failure in general practice are complex. We recommend further research to establish whether multifaceted intervention programmes based on our findings can improve the management of heart failure in primary care.

Pre-clinical safety and efficacy of TA-CIN, a recombinant HPV16 L2E6E7 fusion protein vaccine, in homologous and heterologous prime-boost regimens.

Human papillomavirus (HPV) E6 and E7 oncoproteins are attractive targets for T-cell-based immunotherapy of cervical intraepithelial neoplasia (CIN) and cancer. A newly designed vaccine, comprising the HPV16 L2, E6 and E7 as a single fusion protein (TA-CIN), was shown to elicit HPV16-specific CTL, T-helper cells and antibodies in a pre-clinical mouse model. These immune responses effectively prevented outgrowth of HPV16-positive tumour cells in a prophylactic setting as well as in a minimal residual disease setting. CTL immunity was optimally induced when TA-CIN was employed in heterologous prime-boost regimens in combination with TA-HPV, a clinical grade vaccinia-based vaccine. These data provide a scientific basis for the use of TA-CIN, alone or in combination with TA-HPV in future human trials.

Measuring human T-lymphocyte function.

lymphocytes (T cells) play critical roles in the regulation of immune responses, and are responsible for mediating many of the effector mechanisms of the immune system. For this reason, there has always been a need for assays to measure accurately the activity of populations of T cells, both in model (animal) systems and in humans. The expansion of the biotechnology industry has led to a dramatic increase in the number of novel immunotherapeutics that are being developed for the treatment of cancer, autoimmune disorders and infectious diseases. This increase in activity in the field of immunotherapy, coupled with the expense of clinical trials, has led to renewed interest in methods that accurately assess T-cell function, as researchers seek to maximise the amount of information that can be obtained from each clinical study. Assessing the quantitative and qualitative nature of a T-cell response, for example following vaccination or immunosuppressive therapy, can provide valuable information about the efficacy of a treatment, in place of a clinical endpoint. This article reviews some of the established methods that are used to monitor human T-cell activity, and describes some new approaches that are in development to increase the speed, sensitivity and relevance of such methods.

Construction and characterisation of a recombinant vaccinia virus expressing human papillomavirus proteins for immunotherapy of cervical cancer.

The presence and consistent expression of the genes encoding the human papillomavirus (HPV) E6 and E7 proteins in the great majority of cervical tumours presents the opportunity for an immunotherapeutic approach for control of the disease. This report describes the construction and characterisation of a recombinant vaccinia virus designed to express modified forms of the E6 and E7 proteins from HPV16 and HPV18, the viruses most commonly associated with cervical cancer. The recombinant virus (designated TA-HPV) was based on the Wyeth vaccine strain of vaccinia, and was shown to express the desired gene products. Studies in mice indicated that the recombinant virus was less neurovirulent than the parental virus and was capable of inducing an HPV-specific CTL response. This pre-clinical evaluation has provided a basis for the initiation of human trials in cervical cancer patients.

A recombinant vaccinia virus encoding human papillomavirus types 16 and 18, E6 and E7 proteins as immunotherapy for cervical cancer.

BACKGROUND: Human papillomavirus (HPV) infection, especially with type 16 or 18, is associated with cervical cancer. Two HPV proteins, E6 and E7, are consistently expressed in tumour cells. The objectives of the study were to examine the clinical and environmental safety and immunogenicity in the first clinical trial of a live recombinant vaccinia virus expressing the E6 and E7 proteins of HPV 16 and 18 (TA-HPV). METHODS: The study was an open label phase I/II trial in eight patients with late stage cervical cancer. The patients were vaccinated with a single dose of TA-HPV and kept in strict isolation to monitor local and systemic side-effects, environmental spread, and anti-E6/E7 immune responses. FINDINGS: Vaccination resulted in no significant clinical side-effects and there was no environmental contamination by live TA-HPV. Each patient mounted an antivaccinia antibody response and three of the eight patients developed an HPV-specific antibody response that could be ascribed to the vaccination. HPV-specific cytotoxic T lymphocytes, the effector mechanism most likely to be of therapeutic benefit, were detected in one of three evaluable patients. INTERPRETATION: Further studies to investigate the use ot TA-HPV for immunotherapy of cervical cancer are warranted.

Human cytotoxic T lymphocytes stimulated by endogenously processed human papillomavirus type 11 E7 recognize a peptide containing a HLA-A2 (A*0201) motif.

Cytotoxic T lymphocytes (CTL) may play an important role in the control of human papillomavirus (HPV)-induced anogenital neoplasias, but have been difficult to study owing to the difficulty in obtaining sufficient quantities of infectious virus. To address this we have stimulated human HPV-specific CTL in vitro using low-density cells (LDC) from peripheral blood mononuclear cells (PBMC). Low-density cells were used to present synthetic peptides, or endogenously processed peptides expressed from recombinant vaccinia viruses, to high-density PBMC (predominantly lymphocytes) for 6 days. Cytotoxic T lymphocytes stimulated with endogenously processed HPV 11 E7 recognized the synthetic HLA-A2 (A*0201) motif-containing nonamer, 4-12. In reciprocal experiments, CTL stimulated with this peptide in vitro recognized targets expressing endogenously processed E7. The responses in each case were A2 restricted and peptide specific. Two additional A2 motif-containing nonamers from HPV 6b E7 (21-30 and 47-55) also elicited peptide-specific, A2-restricted CTL. The data illustrate the potential that in vitro stimulation with LDC has in understanding CTL responses to experimentally problematic viral systems such as HPV, and may offer a route to specific immunotherapy of HPV-associated lesions.

Strategies for studying mouse and human immune responses to human papillomavirus type 16.

Cytotoxic T lymphocytes (CTL) are an important protective mechanism in viral infection and can be effective against tumours. We have investigated the tumour-associated E6 and E7 genes of human papillomavirus type 16 as CTL targets. In H-2b mice we have defined epitopes in E6 and E7 which can readily generate CTL in vivo and we have shown that HLA-A2.1 transgenic mice can generate an HLA-A2.1-restricted response. We have been unable to reveal a primed CTL response in humans. These paradoxical findings imply that human papillomavirus may fail to stimulate a systemic CTL response and/or employ strategies for evading or down-regulating such a response.

A method for rapid screening of recombinant proteins for recognition by T lymphocytes.

A simple, cost-effective method is described that allows rapid screening of recombinant protein sequences for their ability to stimulate T cells. Individual microcultures of E. coli each expressing a gene product or peptide sequence fused to protein A are grown in 96-well plates. Following lysis of the bacteria, the fusion peptide is readily captured with immobilized immunoglobulin in tissue culture wells. No further purification is required. T lymphocytes plus appropriate antigen-presenting cells are added directly to the wells and assayed for proliferation. The DNA in bacteria from wells stimulating T cell proliferation is then sequenced. The technique allows rapid mapping of T cell epitopes by facilitating screening of truncation mutants without extensive purification. Described here is a further application of the technique to study monosubstituted analogues of a known T cell epitope.

Human T cell responses to human papillomavirus type 16 L1 and E6 synthetic peptides: identification of T cell determinants, HLA-DR restriction and virus type specificity.

Four T cell determinants in the major capsid protein of human papillomavirus (HPV) type 16 L1 and one in the E6 protein associated with cellular transformation were defined using synthetic peptides to stimulate peripheral blood mononuclear cells from asymptomatic individuals. HLA-DR restriction was defined using murine L cells transfected with HLA-DR genes to present antigen. Responses to two of the five determinants by T cell lines and clones were shown to be specific for HPV-16 based on the lack of cross-recognition of the corresponding sequences of other known papillomavirus sequences (types 1a, 5, 6b, 8, 11, 18 and 33). The T cells raised against two of the other peptides cross-reacted with corresponding peptides from other strains to varying extents, depending on their structural homology. The implications of these results regarding the prevalence of HPV-16 infection in the population and the possible diagnostic role of these responses in papillomavirus infection is discussed.

Polyclonal in vitro proliferative responses from nonimmune donors to Plasmodium falciparum malaria antigens require UCHL1+ (memory) T cells.

The in vitro polyclonal proliferative responses of peripheral blood mononuclear cells to whole blood stage parasites or fractionated antigens from the human malaria parasite Plasmodium falciparum were studied. Cells from healthy laboratory donors who had never been exposed to malaria antigens in vivo consistently proliferated to P. falciparum antigens, as did cord blood mononuclear cells. This response was only observed in sheep rosette-positive cells in the presence of adherent cells and was inhibited by NH4Cl, indicating a requirement for antigen processing. The proliferative response was strongest at day 6 and was dependent on the presence of cells expressing high levels of CD45 180-kD isomer (UCHL1 monoclonal antibody), a marker for activated or memory cells, but not for CD45R (SN130 monoclonal antibody) a marker for naive or unprimed T cells. This suggests a similarity to the recall response to tuberculin antigen. These results suggest that the proliferative response to malaria antigens observed previously and described as a nonspecific mitogenic response may be a cross-reactive response to epitopes shared between P. falciparum and other common immunogens. This would explain the establishment of T cell clones to malaria antigens from such donors, but might suggest that the epitopes to which such clones are specific may be of questionable protective or diagnostic use.

Peptides recognized by class I restricted T cells also bind to MHC class II molecules.

The mechanisms of antigen recognition employed by both class I and class II MHC-restricted T cells are very similar, yet many of the T cell determinants described to date are recognized in the context of a single class of MHC molecules, and generally with only one or a very few different MHC alleles. To determine whether this might be due to a structural difference between class I and class II restricted T cell determinants, peptides previously shown to be recognized in the context of MHC class I proteins by mouse or human CD8+ T lymphocytes were tested for their capacity to bind to HLA-DR molecules on the surface of B lymphoblastoid cell lines (B-LCL). Four out of five class I restricted T cell determinants tested bound to a panel of B-LCL, and the binding was inhibited by anti-HLA-DR mAb. The peptides did not bind to the class II-negative B-LCL RJ2.2.5 nor to mouse L cells, but did bind to L cells transfected with HLA-DR1.