Chronic stress alters the immune response to influenza virus vaccine in older adults.

To determine whether a chronic stressor (caregiving for a spouse with a progressive dementia) is associated with an impaired immune response to influenza virus vaccination, we compared 32 caregivers' vaccine responses with those of 32 sex-, age-, and socioeconomically matched control subjects. Caregivers showed a poorer antibody response following vaccination relative to control subjects as assessed by two independent methods, ELISA and hemagglutination inhibition. Caregivers also had lower levels of in vitro virus-specific-induced interleukin 2 levels and interleukin 1beta; interleukin 6 did not differ between groups. These data demonstrate that down-regulation of the immune response to influenza virus vaccination is associated with a chronic stressor in the elderly. These results could have implications for vulnerability to infection among older adults.

A Schistosoma mansoni fatty acid-binding protein, Sm14, is the potential basis of a dual-purpose anti-helminth vaccine.

Molecular cloning of components of protective antigenic preparations has suggested that related parasite fatty acid-binding proteins could form the basis of the protective immune crossreactivity between the parasitic trematode worms Fasciola hepatica and Schistosoma mansoni. Molecular models of the two parasite proteins showed that both molecules adopt the same basic three-dimensional structure, consisting of a barrel-shaped molecule formed by 10 antiparallel beta-pleated strands joined by short loops, and revealed the likely presence of crossreactive, discontinuous epitopes principally derived from amino acids in the C-terminal portions of the molecules. A recombinant form of the S. mansoni antigen, rSm14, protected outbred Swiss mice by up to 67% against challenge with S. mansoni cercariae in the absence of adjuvant and without provoking any observable autoimmune response. The same antigen also provided complete protection against challenge with F. hepatica metacercariae in the same animal model. The results suggest that it may be possible to produce a single vaccine that would be effective against at least two parasites, F. hepatica and S. mansoni, of veterinary and human importance, respectively.

Production of infectious human respiratory syncytial virus from cloned cDNA confirms an essential role for the transcription elongation factor from the 5' proximal open reading frame of the M2 mRNA in gene expression and provides a capability for vaccine development.

Infectious human respiratory syncytial virus (RSV) was produced by the intracellular coexpression of five plasmid-borne cDNAs. One cDNA encoded a complete positive-sense version of the RSV genome (corresponding to the replicative intermediate RNA or antigenome), and each of the other four encoded a separate RSV protein, namely, the major nucleocapsid N protein, the nucleocapsid P phosphoprotein, the major polymerase L protein, or the protein from the 5' proximal open reading frame of the M2 mRNA [M2(ORF1)]. RSV was not produced if any of the five plasmids was omitted. The requirement for the M2(ORF1) protein is consistent with its recent identification as a transcription elongation factor and confirms its importance for RSV gene expression. It should thus be possible to introduce defined changes into infectious RSV. This should be useful for basic studies of RSV molecular biology and pathogenesis; in addition, there are immediate applications to the development of live attenuated vaccine strains bearing predetermined defined attenuating mutations.

Development of a safe live Leishmania vaccine line by gene replacement.

Vaccination with live Leishmania major has been shown to yield effective immunization in humans; however, this has been discontinued because of problems associated with virulence of the available vaccine lines. To circumvent this, we tested the ability of a dhfr-ts- null mutant of L. major, obtained by gene targeting, to infect and then to vaccinate mice against challenge with virulent L. major. Survival and replication of dhfr-ts- in macrophages in vitro were dependent upon thymidine, with parasites differentiating into amastigotes prior to destruction. dhfr-ts- parasites persisted in BALB/c mice for up to 2 months, declining with a half-life of 2-3 days. Nonetheless, dhfr-ts- was incapable of causing disease in both susceptible and immunodeficient (nu/nu) BALB/c mice. Animal infectivity could be partially restored by thymidine supplementation. When inoculated by the i.v., s.c., or i.m. routes into mice, dhfr-ts- could elicit substantial resistance to a subsequent challenge with virulent L. major. Thus, Leishmania bearing auxotrophic gene knockouts can be safe and induce protective immunity. Potentially, dhfr-ts- could be used as a platform for delivery of immunogens relevant to other diseases.

Minimal epitopes expressed in a recombinant polyepitope protein are processed and presented to CD8+ cytotoxic T cells: implications for vaccine design.

The epitopes recognized by CD8+ cytotoxic T lymphocytes (CTL) are generated from cytosolic proteins by proteolytic processing. The nature of the influences exerted by the sequences flanking CTL epitopes on these processing events remains controversial. Here we show that each epitope within an artificial polyepitope protein containing nine minimal CD8+ CTL epitopes in sequence was processed and presented to appropriate CTL clones. Natural flanking sequences were thus not required to direct class I proteolytic processing. In addition, unnatural flanking sequences containing other CTL epitopes did not interfere with processing. The ability of every CTL epitope to be effectively processed from a protein containing only CTL epitopes is likely to find application in the construction of recombinant polyepitope CTL vaccines.

Recombinant Listeria monocytogenes as a live vaccine vehicle for the induction of protective anti-viral cell-mediated immunity.

Listeria monocytogenes (LM) is a Gram-positive bacterium that is able to enter host cells, escape from the endocytic vesicle, multiply within the cytoplasm, and spread directly from cell to cell without encountering the extracellular milieu. The ability of LM to gain access to the host cell cytosol allows proteins secreted by the bacterium to efficiently enter the pathway for major histocompatibility complex class I antigen processing and presentation. We have established a genetic system for expression and secretion of foreign antigens by recombinant strains, based on stable site-specific integration of expression cassettes into the LM genome. The ability of LM recombinants to induce protective immunity against a heterologous pathogen was demonstrated with lymphocytic choriomeningitis virus (LCMV). LM strains expressing the entire LCMV nucleoprotein or an H-2Ld-restricted nucleoprotein epitope (aa 118-126) were constructed. Immunization of mice with LM vaccine strains conferred protection against challenge with virulent strains of LCMV that otherwise establish chronic infection in naive adult mice. In vivo depletion of CD8+ T cells from vaccinated mice abrogated their ability to clear viral infection, showing that protective anti-viral immunity was due to CD8+ T cells.

Durable complete clinical responses in a phase I/II trial using an autologous melanoma cell/dendritic cell vaccine

Advanced metastatic melanoma is incurable by standard treatments, but occasionally responds to immunotherapy. Recent trials using dendritic cells (DC) as a cellular adjuvant have concentrated on defined peptides as the source of antigens, and rely on foreign proteins as a source of help to generate a cell-mediated immune response. This approach limits patient accrual, because currently defined, non-mutated epitopes are restricted by a small number of human leucocyte antigens. It also fails to take advantage of mutated epitopes peculiar to the patient's own tumour, and of CD4(+) T lymphocytes as potential effectors of anti-tumour immunity. We therefore sought to determine whether a fully autologous DC vaccine is feasible, and of therapeutic benefit. Patients with American Joint Cancer Committee stage IV melanoma were treated with a fully autologous immunotherapy consisting of monocyte-derived DC, matured after culture with irradiated tumour cells. Of 19 patients enrolled into the trial, sufficient tumour was available to make treatments for 17. Of these, 12 received a complete priming phase of six cycles of either 0.9X10(6) or 5X10(6) DC/intradermal injection, at 2-weekly intervals. Where possible, treatment continued with the lower dose at 6-weekly intervals. The remaining five patients could not complete priming, due to progressive disease. Three of the 12 patients who completed priming have durable complete responses (average duration 3 5 months +), three had partial responses, and the remaining six had progressive disease (WHO criteria). Disease regression was not correlated with dose or with the development of delayed type hypersensitivity responses to intradermal challenge with irradiated, autologous tumour. However, plasma S-100B levels prior to the commencement of treatment correlated with objective clinical response (P = 0.05) and survival (log rank P < 0.001). The treatment had minimal side-effects and was well tolerated by all patients. Mature, monocyte-derived DC preparations exposed to appropriate tumour antigen sources can be reliably produced for patients with advanced metastatic melanoma, and in a subset of those patients with lower volume disease their repeated administration results in durable complete responses.

Studies of maternal immunisation with pneumococcal polysaccharide vaccine in Papua New Guinea

two studies, pneumococcal polysaccharide (Pnc PS) vaccine was given to more than 400 pregnant Papua New Guinean women. No deleterious effects were found. The vaccine prevented acute lower respiratory infection (ALRI) among offspring in utero or aged 1-17 months at the time of maternal immunisation, suggesting protection through breast feeding. Serum IgG antibody titres were higher in vaccinated than unvaccinated groups for 2-4 months after delivery and no immune suppression, evaluated by the response to subsequent Pnc PS vaccination, was detected. Breast milk IgA to four serotypes was 1.1-1.8 times higher in immunised than unimmunised women for 6 months postpartum. Given results from several developing countries, large-scale safety and efficacy trials are now justified. Postpartum maternal immunisation is another intervention under consideration. (C) 2003 Elsevier Science Ltd. All rights reserved.

HPV-16 L1 VLP vaccine elicits a broad-spectrum of cytokine responses in whole blood

Here, we evaluated innate and adaptive immune system cytokine responses induced by HPV-16 L1 VLP in whole blood (WB) cultures from individuals receiving the vaccine (n = 20) or placebo (n = 4) before and after vaccination. 11 cytokines were measured: IL- 1 beta, IL-2, IL-4, IL-5, IL-6, IL-8, 1L- 10, IL- 12, IFN-gamma, TNF-alpha, and GM-CSF using multiplex bead arrays. Cytokine profiles from WB samples clearly discriminated between vaccine and placebo recipients and between pre and post-vaccination responses. Significant increases in Th1, Th2 and inflammatory cytokines were observed in WB assays following vaccination. Results from WB assays were compared against parallel PBMC-based assays in a subset of patients. Differences between whole blood assay and PBMC were observed, with the highest levels of induction found for WB for several cytokines. Our results indicate that multiplex assays for cytokine profiling in WB are an efficient toot for assessing broad spectrum, innate and adaptive immune responses to vaccines and identifying immunologic correlates of protection in efficacy studies. (c) 2005 Elsevier Ltd. All rights reserved.

A polytope DNA vaccine elicits multiple effector and memory CTL responses and protects against human papillomavirus 16 E7-expressing tumour

Vaccine-induced CD8 T cells directed to tumourspecific antigens are recognised as important components of protective and therapeutic immunity against tumours. Where tumour antigens have pathogenic potential or where immunogenic epitopes are lost from tumours, development of subunit vaccines consisting of multiple individual epitopes is an attractive alternative to immunising with whole tumour antigen. In the present study we investigate the efficacy of two DNA-based multiepitope('polytope') vaccines containing murine (H-2(b)) and human (HLA-A* 0201)-restricted epitopes of the E7 oncoprotein of human papillomavirus type 16, in eliciting tumour-protective cytotoxic T-lymphocyte (CTL) responses. We show that the first of these polytopes elicited powerful effector CTL responses ( measured by IFN-gamma ELISpot) and long-lived memory CTL responses ( measured by functional CTL assay and tetramers) in immunised mice. The responses could be boosted by immunisation with a recombinant vaccinia virus expressing the polytope. Responses induced by immunisation with polytope DNA alone partially protected against infection with recombinant vaccinia virus expressing the polytope. Complete protection was afforded against challenge with an E7-expressing tumour, and reduced growth of nascent tumours was observed. A second polytope differing in the exact composition and order of CTL epitopes, and lacking an inserted endoplasmic reticulum targeting sequence and T-helper epitope, induced much poorer CTL responses and failed to protect against tumour challenge. These observations indicate the validity of a DNA polytope vaccine approach to human papillomavirus E7 - associated carcinoma, and underscore the importance of design in polytope vaccine construction.

Delipidation of a hepadnavirus: Viral inactivation and vaccine development

Many viruses including HIV, hepatitis C and hepatitis B, have an outer lipid envelope which maintains inserted viral peptides in the “correct” functional conformation and orientation. Disruption of the lipid envelope by most solvents destroys infectivity and often results in a loss of antigenicity. This communication outlines a novel approach to viral inactivation by specific solvent delipidation which modifies the whole virion rendering it non-infective, but antigenic. Duck hepatitis B virus (DHBV) was delipidated using a diisopropylether (DIPE) and butanol mixture and residual infectivity tested by inoculation into day-old ducks. Delipidation completely inactivated the DHBV (p < 0.001). Delipidated DHBV was then used to vaccinate ducks. Three doses of delipidated DHBV induced anti-DHBs antibody production and prevented high dose challenge infection in five out of six ducks. In comparison, five of six ducks vaccinated with undelipidated DHBV and four of four ducks vaccinated with glutaraldehyde inactivated DHBV were unprotected (p < 0.05). Although this solvent system completely inactivated DHBV, viral antigens were retained in an appropriate form to induce immunity. Delipidation of enveloped viruses with specific organic solvents has potential as the basis for development of vaccines.