Enhanced immunogenicity for CD8+ T cell induction and complete protective efficacy of malaria DNA vaccination by boosting with modified vaccinia virus Ankara.

Immunization with irradiated sporozoites can protect against malaria infection and intensive efforts are aimed at reproducing this effect with subunit vaccines. A particular sequence of subunit immunization with pre-erythrocytic antigens of Plasmodium berghei, consisting of single dose priming with plasmid DNA followed by a single boost with a recombinant modified vaccinia virus Ankara (MVA) expressing the same antigen, induced unprecedented complete protection against P. berghei sporozoite challenge in two strains of mice. Protection was associated with very high levels of splenic peptide-specific interferon-gamma-secreting CD8+ T cells and was abrogated when the order of immunization was reversed. DNA priming followed by MVA boosting may provide a general immunization regime for induction of high levels of CD8+ T cells.

Severe pneumonia and jaundice in a young man: an atypical presentation of an uncommon disease.

We present a patient with an atypical presentation of Fusobacterium infection, the genus responsible for Lemierre's syndrome. This syndrome, which often affects healthy, young people and can be fatal if not recognized and treated early, is defined as a history of recent oropharyngeal infection with clinical or radiological evidence of internal jugular vein thrombosis and isolation of anaerobic pathogens, mainly Fusobacterium necrophorum. The history, presentation, investigations and management of the patient are described and then contrasted with the existing literature surrounding Lemierre's syndrome, once termed the 'forgotten disease'.

Chlamydial infections.

Chlamydiae are among the most successful bacterial pathogens, and there are few branches of medicine on which chlamydial infection and its sequelae do not impinge. Chlamydia trachomatis is responsible for many million cases of blindness, pelvic inflammatory disease, urethritis, epididymitis, infertility and ectopic pregnancy annually; it also causes lymphogranuloma venereum, reactive arthritis, ophthalmia neonatorum and infantile pneumonia. C. pneumoniae is among the most common causes of community-acquired pneumonia, and recent evidence suggests that it may play a part in the pathogenesis of coronary heart disease. C. psittaci is a highly prevalent zoonotic infection with a wide host range. It is of great economic importance, and causes sporadic but sometimes devastating disease in humans. Most chlamydial infections are subclinical, but even if the initial illness is mild there may be serious long-term sequelae. It is therefore important to identify and treat chlamydial infections in their early stages, but diagnosis usually depends on laboratory tests. Recent trials have shown that single doses of the long-acting macrolide azithromycin are effective in the treatment of genital and ocular C. trachomatis infection, but longer courses of antimicrobials remain the mainstay of treatment for C. pneumoniae and C. psittaci infections.

Recipient characteristics in the transduction of methicillin resistance in Staphylococcus epidermidis.

Methicillin resistance (Mecr) was transduced into a methicillin-susceptible variant of the Mecr donor Staphylococcus epidermidis BS10. UV irradiation of phage stimulated Mecr transduction frequency. If loss of Cd and Hg ion resistance occurred in this recipient, or if the three markers Mecr, Cdr, and Hgr were co-eliminated from the donor, neither strain acted as a recipient for Mecr.

Modified vaccinia virus Ankara undergoes limited replication in human cells and lacks several immunomodulatory proteins: implications for use as a human vaccine.

Modified virus Ankara (MVA) is a vaccinia virus (VV) strain that was attenuated by serial passage through chick embryo fibroblasts (CEFs) and contains six large genomic deletions compared with parental virus. MVA replicates well in CEFs, but poorly in most mammalian cells. Recombinant MVA is a promising human vaccine candidate due to its restricted host range, immunogenicity and avirulence in animal models, and excellent safety record as a smallpox vaccine. Here we present a further characterization of MVA and demonstrate that: (i) MVA can replicate, albeit poorly, in transformed human cell lines, but not in primary human fibroblasts although there is limited cell-to-cell spread; (ii) MVA is a potent inducer of type I interferon (IFN) from primary human cells, which may restrict virus spread in vivo; and (iii) unlike other VV strains, MVA does not express soluble receptors for IFN-gamma, IFN-alpha/beta, tumour necrosis factor and CC chemokines, but does express a soluble interleukin-1beta receptor. This provides a plausible and testable explanation for the good immunogenicity of MVA despite its poor replication in mammals. The implications of these findings for the use of MVA as a safe and immunogenic human vaccine candidate are discussed.

Effective induction of HIV-specific CTL by multi-epitope using gene gun in a combined vaccination regime.

Reliable and effective induction of cytotoxic T-lymphocytes (CTL) is one of the prime objectives of vaccine research. Previously, novel HIV vaccine candidates were constructed as a string of CTL epitopes (20 human, 3 macaque and 1 mouse) delivered using a DNA vector [Hanke T, Schneider J, Gilbert SG, Hill AVS, McMichael A. DNA multi-CTL epitope vaccines for HIV and Plasmodium falciparum: immunogenicity in mice. Vaccine 1998;16:426-435.] or modified vaccinia Ankara (MVA [Hanke T, Blanchard TJ, Schneider J, Ogg GS, Tan R, Becker MSC, Gilbert SG, Hill AVS, Smith GL, McMichael A. Immunogenicities of intravenous and intramuscular administrations of MVA-based multi-CTL epitope vaccine for HIV in mice. J Gen Virol 1998;79:83-90.]), i.e. vaccine vehicles acceptable for use in humans. In mice, a single intramuscular (i.m.) needle injection of either vaccine alone elicited good CTL responses. Here, it is demonstrated that the multi-epitope DNA also induced CTL when delivered intradermally using the Accell gene gun. The CTL responses increased after re-immunization and after three deliveries were comparable to those induced by a single i.m. injection. Recent evidence indicates that combining routes and vaccine vehicles enhances the immunogenicity of vaccine-delivered or -encoded antigens. Here, it is shown that administration of DNA by an i.m. priming/gene gun boosting more efficiently induced CTL than gene gun priming/i.m. boosting. A similar increment was obtained by sequential vaccinations using a gene gun-delivered DNA followed by recombinant MVA. Thus particular sequences of routes or vaccine vehicles rather than simple prime-boost delivery of a single vaccine is critical for an effective elicitation of CTL.

Induction of CD8+ T-lymphocyte responses to a secreted antigen of Mycobacterium tuberculosis by an attenuated vaccinia virus.

Protective immunity against Mycobacterium tuberculosis infection requires the activation of mycobacterium-specific CD8+ T cells, as well as CD4+ T cells. Therefore, optimizing strategies that stimulate CD8+ T cells recognizing dominant mycobacterial antigens, including secreted proteins, may lead to the development of more effective vaccines against tuberculosis. To generate a viral vaccine that is safe in humans, the early secreted protein, MPT64, was expressed in the attenuated vaccinia virus (VV) strain, modified vaccinia virus Ankara (MVA-64). The immunogenicity of MVA-64 was compared with that of the Western Reserve strain of VV (VVWR-64). The replication-defective MVA-64 was as efficient as VVWR-64 in inducing specific antibodies and cytolytic T-cell responses to a defined H-2-Db-restricted epitope on MTP-64. In addition, priming with MPT64-expressing plasmid DNA (DNA-64), and boosting with either MVA-64 or VVWR-64, markedly enhanced MPT64-specific cytolytic and IFN-gamma-producing CD8+ T-cell responses. These findings suggest that MVA may be a suitable vaccine carrier for stimulating mycobacterium-specific CD8+ T-cell responses and may be particularly relevant for developing vaccines for use in regions endemic for tuberculosis and HIV infection.

Ty virus-like particles, DNA vaccines and Modified Vaccinia Virus Ankara; comparisons and combinations.

Three types of vaccine, all expressing the same antigen from Plasmodium berghei, or a CD8+ T cell epitope from that antigen, were compared for their ability to induce CD8+ T cell responses in mice. Higher levels of lysis and numbers of IFN-gamma secreting T cells were primed with Ty virus-like particles and Modified Vaccinia Virus Ankara (MVA) than with DNA vaccines, but none of the vaccines were able to protect immunised mice from infectious challenge even after repeated doses. However, when the immune response was primed with one type of vaccine (Ty-VLPs or DNA) and boosted with another (MVA) complete protection against infection was achieved. Protection correlated with very high levels of IFN-gamma secreting T cells and lysis. This method of vaccination uses delivery systems and routes that can be used in humans and could provide a generally applicable regime for the induction of high levels of CD8+ T cells.

Immunogenicities of intravenous and intramuscular administrations of modified vaccinia virus Ankara-based multi-CTL epitope vaccine for human immunodeficiency virus type 1 in mice.

A vaccine against human immunodeficiency virus (HIV) is still awaited. Although the correlates of protection remain elusive, it is likely that CD8+ T cells play an important role in the control of this infection. To firmly establish the importance of these cells in protective immunity, a means of efficient elicitation of CD8+ T cell responses in the absence of antibody is needed and, when available, might represent a crucial step towards a protective vaccine. Here, a novel vaccine candidate was constructed as a multi-cytotoxic T lymphocyte (CTL) epitope gene delivered and expressed using modified vaccinia virus Ankara (MVA). The immunogen consists of 20 human, one murine and three rhesus macaque epitopes. The non-human epitopes were included so that the vaccine can be tested for immunogenicity and optimal vaccination doses, routes and regimes in experimental animals. Mice were immunized intravenously (i.v.) or intramuscularly (i.m.) using a single dose of 10(6) p.f.u. of the recombinant MVA and the induction of CTL was assessed. It was demonstrated that both administration routes induced specific CTL responses and that the i.v. route was moderately more immunogenic than the i.m. route. The frequencies of ex vivo splenocytes producing interferon-y upon MHC class I-restricted peptide stimulation were determined using an ELISPOT assay. Also, the correct processing and presentation of some HLA-restricted epitopes in human cells was confirmed.

Enhancement of MHC class I-restricted peptide-specific T cell induction by a DNA prime/MVA boost vaccination regime.

Human immunodeficiency virus (HIV) vaccine candidates were previously constructed as a string of cytotoxic T lymphocyte (CTL) epitopes delivered and expressed using DNA and modified virus Ankara (MVA; an attenuated vaccinia virus) vectors. These vaccines were shown to induce interferon (IFN)-gamma-producing and cytolytic CD8+ T cells after a single vaccine administration. In the course of this work, immunization protocols were sought which would improve the levels of induced HIV-specific T cells. It was found that previous immunological exposure to MVA reduced the efficiency of subsequent priming and boosting using the same vaccine vehicle. However, a combined regime whereby the animals were first primed with the DNA vaccine and then boosted with MVA was the most potent protocol for the induction of both interferon-gamma-producing and cytolytic T cells against two CTL epitopes simultaneously. The general applicability of this novel vaccination method for induction of major histocompatibility complex class I-restricted T cells is discussed.

Synthetic peptides based on Chlamydia trachomatis antigens identify cytotoxic T lymphocyte responses in subjects from a trachoma-endemic population.

CD8+ cytotoxic T lymphocytes (CTL) recognize peptide antigens in the context of class I MHC antigen molecules. To identify peptides capable of eliciting anti-Chlamydia trachomatis CTL responses, 13 synthetic peptides conforming to human leucocyte antigen (HLA)-B8- or -B35-predicted binding motifs were synthesized using sequences based on C. trachomatis major outer membrane protein (MOMP) and heat shock protein 60 (hsp60). Two of 11 HLA-B35-predicted binding peptides were able to stabilize HLA-B35 in an in vitro binding assay. All peptides were tested in CTL assays using peripheral blood mononuclear cells (PBMC) isolated from 26 HLA-B8 or -B35 individuals resident in a trachoma-endemic community. Responses to MOMP and hsp60 peptides were identified in a minority of both HLA-B8 and -B35 individuals. Two of 12 HLA-B8 subjects responded to MOMP and 1/13 to hsp60 peptides. Responses in HLA-B35 subjects were similar, 1/13 subjects responding to MOMP and 2/13 to hsp60 peptides. CTL responses were observed only in children resolving current infection and in adults without scarring of the conjunctiva. These results suggest that anti-chlamydial CTL occur at low levels in peripheral blood, but may be important in the resolution of naturally acquired human ocular chlamydial infection.

Recombinant modified vaccinia virus Ankara efficiently restimulates human cytotoxic T lymphocytes in vitro.

The immunogenicity of recombinant modified vaccinia Ankara, a highly attenuated vaccinia virus, expressing influenza nucleoprotein (MVA-NP) and HIV-1 gag (MVA-gag) was investigated. Restimulation of peripheral blood lymphocytes of healthy subjects with MVA-NP led to expansion of CTL with specificity for known NP epitopes. These CTL efficiently lysed NP peptide-pulsed targets and released interferon-gamma (IFN-gamma) on contact with epitope peptide. MVA-NP-stimulated CTL specific for the HLA-B8 epitope, NP380-88, stained with a tetrameric complex of HLA-B8 refolded with the NP380-88 peptide and anti-CD8 antibody on flow cytometry. CTL were also elicited from two HIV-1 seropositive donors by restimulation with MVA-HIV-1 gag and showed specificity for immunodominant gag epitopes. These data indicate that restimulation of human CTL with recombinant MVA is effective and suggest that MVA will elicit CTL responses in humans in vivo.

Effective induction of simian immunodeficiency virus-specific cytotoxic T lymphocytes in macaques by using a multiepitope gene and DNA prime-modified vaccinia virus Ankara boost vaccination regimen.

DNA and modified vaccinia virus Ankara (MVA) are vaccine vehicles suitable and safe for use in humans. Here, by using a multicytotoxic T-lymphocyte (CTL) epitope gene and a DNA prime-MVA boost vaccination regimen, high levels of CTLs specific for a single simian immunodeficiency virus (SIV) gag-derived epitope were elicited in rhesus macaques. These vaccine-induced CTLs were capable of killing SIV-infected cells in vitro. Fluorescence-activated cell sorter analysis using soluble tetrameric major histocompatibility complex-peptide complexes showed that the vaccinated animals had 1 to 5% circulating CD8(+) lymphocytes specific for the vaccine epitope, frequencies comparable to those in SIV-infected monkeys. Upon intrarectal challenge with pathogenic SIVmac251, no evidence for protection was observed in at least two of the three vaccinated animals. This study does not attempt to define correlates of protective immunity nor design a protective vaccine against immunodeficiency viruses, but it demonstrates clearly that the DNA prime-MVA boost regimen is an effective protocol for induction of CTLs in macaques. It also shows that powerful tools for studying the role of CTLs in the control of SIV and human immunodeficiency virus infections are now available: epitope-based vaccines, a protocol for an effective induction of CTLs in primates, and a simple and sensitive method for quantitation of epitope-specific T cells. The advantages of the DNA prime-MVA boost regimen as well as the correlations of tetramer staining of peripheral blood lymphocytes with CTL killing in vitro and postchallenge control of viremia are discussed.

A prime-boost immunisation regimen using DNA followed by recombinant modified vaccinia virus Ankara induces strong cellular immune responses against the Plasmodium falciparum TRAP antigen in chimpanzees.

Two chimpanzees were vaccinated intramuscularly against malaria using plasmid DNA expressing the pre-erythrocytic antigens thrombospondin related adhesion protein (PfTRAP) and liver stage specific antigen-1 (PfLSA-1) of Plasmodium falciparum together with GM-CSF protein. A recombinant modified vaccinia virus Ankara (MVA) expressing PfTRAP was injected intramuscularly 6 weeks later to boost the immune response. This sequence of antigen delivery induced a specific and long-lasting T cell and antibody response to PfTRAP as detected by ELISPOT assay and ELISA. Antibody responses were detected after four DNA injections, and were boosted by injection of recombinant MVA expressing PfTRAP. Interferon-gamma secreting antigen-specific T cells were detected in both animals, but only after boosting with recombinant MVA. By screening a panel of PfTRAP-derived peptides, an epitope was identified that was recognized by cytotoxic T lymphocytes in one of the chimpanzees studied. T cells specific for this epitope were present in PBMCs and liver-infiltrating lymphocytes at a frequency of between 1 in 200 and 1 in 500. The high immunogenicity of this prime-boost regimen in chimpanzees supports further assessment of this delivery strategy for the induction of protection against P. falciparum malaria in humans.