Heterologous prime-boost immunizations with a virosomal and an alphavirus replicon vaccine.

Heterologous prime-boost immunization strategies in general establish higher frequencies of antigen-specific T lymphocytes than homologous prime-boost protocols or single immunizations. We developed virosomes and recombinant Semliki Forest virus (rSFV) as antigen delivery systems, each capable of inducing strong CTL responses in homologous prime-boost protocols. Here, we demonstrate that a heterologous prime-boost with recombinant Semliki Forest virus (rSFV) encoding a fusion protein of E6 and E7 of human papillomavirus (HPV) type 16 and virosomes containing the HPV16 E7 protein resulted in higher numbers of antigen-specific CTL in mice than homologous protocols. Evasion of vector-specific immunity appeared to play a role in establishing these high frequencies, as coinduction of vector-specific responses during the prime immunization reduced the frequency of antigen-specific CTL after a heterologous booster. However, the high numbers of CTL initially primed by the heterologous protocols did not correlate with enhanced responsiveness to in vitro antigenic stimulation, nor in improved cytolytic activity or antitumor responses in vivo compared to a homologous protocol with rSFV. This lack of correlation could not be explained by changes in numbers of regulatory T cells. However, we observed differences in the frequencies of T cell subsets within the E7-specific CD8(+) T cell population, e.g. higher frequencies of central memory T cells upon homologous immunizations compared to heterologous immunizations. The induction of central memory T cells is crucial for a cancer vaccine as these cells are known to rapidly expand upon recall stimulation. This study demonstrates that the strongly increased number of antigen-specific CTL as induced by heterologous prime-boost immunizations, often used as a proof for the enhanced efficacy of such regimes, does not necessarily equal superior functional antitumor responses.

A rapid single-tube multiplex polymerase chain reaction assay for the seven most prevalent alpha-thalassemia deletions and alphaalphaalpha(anti 3.7) alpha-globin gene triplication.

alpha-Globin gene triplications may exacerbate the alpha chain and beta chain imbalance in beta-thalassemia (beta-thal) and may compensate for the effect of alpha-globin gene deletion in alpha-thal. Identification of an alpha-globin gene triplication is, therefore, valuable in predicting the clinical phenotype of the thalassemias. To be able to detect alpha-globin gene triplications, we have modified an existing multiplex polymerase chain reaction (PCR) assay for the seven most prevalent alpha-globin gene deletions by incorporating two triplication-specific primers and concurrently substituting one of the original primers by a newly designed primer. This modified multiplex PCR assay was evaluated by performing the assay on archival DNA samples and on peripheral blood samples from 163 suspected thalassemia cases. It was found to function properly. Our assay thereby represents the first multiplex PCR assay that can detect both the seven most prevalent alpha-globin gene deletions and the alphaalphaalpha(anti 3.7) alpha-globin gene triplication in a single-tube reaction.

A virosomal immunization strategy against cervical cancer and pre-malignant cervical disease.

In this study, we demonstrate that fusion-active virosomes, containing recombinant human papillomavirus type 16 (HPV16) E7 protein antigen, are capable of inducing a robust class I MHC-restricted cytotoxic T-lymphocyte (CTL) response against HPV-transformed tumour cells in a murine model system. Virosomes are reconstituted viral envelopes, which do not contain the genetic material of the native virus. During the reconstitution process, protein antigens can be encapsulated within the virosomes. In the present study, we used virosomes derived from influenza virus. These virosomes retain the cell binding and membrane fusion characteristics of native influenza virus, and have the capacity to deliver encapsulated antigens to the cytosol of antigen-presenting cells through fusion from within acidic endosomes. After immunization of mice with virosomes containing encapsulated HPV16 E7 protein, the animals developed a strong E7-specific CTL response as assessed by 51Cr release measurements and MHC tetramer staining of spleen cells. Immunization with E7-containing virosomes also resulted in E7-specific antibody responses. In tumour challenge experiments, immunization of mice with E7-containing virosomes prevented tumour outgrowth in >70% of the animals. Thus, influenza-derived virosomes with encapsulated HPV E7 protein antigen act as an excellent vaccine delivery system for induction of cellular immunity against HPV-transformed cells and represent a promising immunotherapeutic vaccine for the treatment of (precursor lesions of) cervical cancer.

Recombinant alphaviruses as vectors for anti-tumour and anti-microbial immunotherapy.

BACKGROUND: Vectors derived from alphaviruses are gaining interest for their high transfection potency and strong immunogenicity. OBJECTIVES: After a brief introduction on alphaviruses and their vectors, an overview is given on current preclinical immunotherapy studies using vector systems based on alphaviruses. The efficacy of alphavirus vectors in inducing immune responses will be illustrated by a more detailed description of immunization studies using recombinant Semliki Forest virus for the treatment of human papilloma virus-induced cervical cancer. RESULTS: Immunization with recombinant alphavirus results in the induction of humoral and cellular immune responses against microbes, infected cells and cancer cells. Preclinical studies demonstrate that infectious diseases and cancer can be treated prophylactically as well as therapeutically. CONCLUSIONS: Alphavirus-based genetic immunization strategies are highly effective in animal model systems, comparing quite favourably with any other approach. Therefore, we hope and expect to see an efficient induction of tumour-or microbial immunity and a positive outcome in future clinical efficacy studies.

Virosomes for antigen and DNA delivery.

Specific targeting and delivery as well as the display of antigens on the surface of professional antigen-presenting cells (APCs) are key issues in the design and development of new-generation vaccines aimed at the induction of both humoral and cell-mediated immunity. Prophylactic vaccination against infectious diseases in general aims at the induction of humoral immune responses to prevent infection. This humoral immune response is mediated by antibody-producing B cells. On the other hand, therapeutic immunisation against virally infected cells and tumour cells requires the induction of cytotoxic T lymphocytes (CTLs) that can specifically recognise and lyse infected cells or transformed tumour cells. The induction of Major Histocompatibility Complex (MHC) class I restricted CTL activity is optimally achieved by synthesis of antigens within APCs, for example, after immunisation with live attenuated virus. However, immunisation with live vaccines bears the risk of causing disease. Therefore, alternative vaccine delivery systems, which enable introduction of nonreplicating antigen into the MHC class I presentation pathway, are sought. Furthermore, for the induction of effective humoral and cellular responses, MHC class II restricted activation of T helper cells (Th cells) is required. Among other delivery systems, as described in this theme issue of Advanced Drug Delivery Reviews, virosomes seem ideally suited for delivery of antigens into both MHC pathways. In this review, we will focus on the use of virosomes as carrier vehicles for the intracellular delivery of protein antigens and DNA, and the induction of a cellular immune response against encapsulated protein antigens and proteins expressed by virosome-associated plasmids.

Leaky cell syndrome: a rare cause of pseudohyperkalaemia.

Life-threatening situations of hyperkalaemia are often caused by renal failure, hyperglycaemia or medication. However pseudohyperkalaemia, a falsely elevated potassium concentration, is usually caused by haemolysis, repeated clenching of the fist during venepuncture or abnormal cell numbers. Another rare cause of pseudohyperkalaemia is familial pseudohyperkalaemia, an autosomal dominantly inherited trait, with increased leakage of potassium from erythrocytes. Under normal in vivo conditions, this increased leakage is compensated by augmented activity of the Na(+)/K(+) ATPase pump. However, after venepuncture the blood cools down to room temperature, reducing the activity of the Na(+)/K(+) ATPase pump whereby the increased potassium leakage becomes more apparent. Here, we present a Dutch patient with extreme familial pseudohyperkalaemia. Interestingly, his two children also show increased potassium leakage at room temperature, albeit at a lower level. Despite the low prevalence of familial pseudohyperkalaemia, it can have important clinical implications and rapid recognition is desired.

Role of T cell competition in the induction of cytotoxic T lymphocyte activity during viral vector-based immunization regimens.

T cell competition between antigen- and vector-specific T cells may determine the outcome of viral vector-based immunization regimens, as we previously proposed. Here, we unravelled the interplay between antigen- and vector-specific immunity, using recombinant Semliki Forest virus (rSFV). Priming of mice with rSFV, encoding LacZ (SFVLacZ), or with empty rSFV strongly suppressed subsequent induction of ovalbumin or Human Papilloma virus (HPV) E6/E7-specific CTL activity by a booster with SFVeOVA or SFVeE6,7, respectively. Yet, priming with irradiated, i.e. replication-defective, SFVLacZ did not affect subsequent CTL induction, indicating that the interfering vector-specific immunity was directed against the viral replicase. However, immune responses against the strongly immunogenic nucleoprotein of influenza virus encoded by SFV were less severely affected by priming with SFVLacZ. Thus the outcome of heterologous prime-boost immunizations appears to depend on the immunogenicity of the respective antigens.

The effect of pre-existing immunity on the capacity of influenza virosomes to induce cytotoxic T lymphocyte activity.

Protein antigens encapsulated in virosomes generated from influenza virus can induce antigen-specific cytotoxic T lymphocyte (CTL) responses. In the present study we determined, in a murine model system, whether pre-existing immunity against influenza virus hampers the induction of a CTL response. CTL induction was only slightly reduced by pre-injection of influenza virus-specific antibodies or pre-exposure to influenza virus. Both pretreatments resulted in the same level of reduction, suggesting that virus-specific antibodies rather than T cell responses account for the reduction. Furthermore, a booster immunization enhanced CTL activation, indicating that virosome-specific immunity induced by a prime immunization does not hamper the booster effect. In conclusion, CTL induction against virosome-encapsulated protein antigens is not significantly inhibited by pre-existing humoral or cellular immunity against influenza virus.

Virosome-mediated delivery of protein antigens in vivo: efficient induction of class I MHC-restricted cytotoxic T lymphocyte activity.

Induction of CTL responses against protein antigens is an important aim in vaccine development. In this paper we present fusion-active virosomes as a vaccine delivery system capable of efficient induction of CTL responses in vivo. Virosomes are reconstituted viral membranes, which do not contain the genetic material of the virus they are derived from. Foreign macromolecules, including protein antigens, can be encapsulated in virosomes during the reconstitution process. Functionally reconstituted virosomes retain the cell binding and fusion characteristics of the native virus. Thus, upon uptake by cells through receptor-mediated endocytosis, virosomes will deliver their content to the cell cytosol. In a previous study, we demonstrated that protein antigens delivered in this manner to dendritic cells are efficiently processed for both MHC class I and class II presentation. Here, we studied in vivo induction of cellular immune responses against virosome-encapsulated ovalbumin (OVA) in mice. As little as 0.75 microg OVA delivered by fusion-active virosomes was sufficient to induce a powerful class I MHC-restricted CTL response. All immunization routes that were used (i.m., i.p. and s.c.) resulted in efficient induction of CTL activity. The CTLs induced were cytotoxic in a standard 51Cr-release assay and produced IFNgamma in response to OVA peptide. Thus, virosomes represent an ideal antigen delivery system for induction of cellular immunity against encapsulated protein antigens.