Tomáš Hanke

Induction of AIDS Virus-Specific CTL Activity in Fresh, Unstimulated Peripheral Blood Lymphocytes from Rhesus Macaques Vaccinated with a DNA Prime/Modified Vaccinia Virus Ankara Boost Regimen

The observed role of CTL in the containment of AIDS virus replication suggests that an effective HIV vaccine will be required to generate strong CTL responses. Because epitope-based vaccines offer several potential advantages for inducing strong, multispecific CTL responses, we tested the ability of an epitope-based DNA prime/modified vaccinia virus Ankara (MVA) boost vaccine to induce CTL responses against a single SIVgag CTL epitope. As assessed using both 51Cr release assays and tetramer staining of in vitro stimulated PBMC, DNA vaccinations administered to the skin with the gene gun induced and progressively increased p11C, C→M (CTPYDINQM)-specific CD8+ T lymphocyte responses in six of six Mamu-A*01+ rhesus macaques. Tetramer staining of fresh, unstimulated PBMC from two of the DNA-vaccinated animals indicated that as much as 0.4% of all CD3+/CD8α+ T lymphocytes were specific for the SIVgag CTL epitope. Administration of MVA expressing the SIVgag CTL epitope further boosted these responses, such that 0.8–20.0% of CD3+/CD8α+ T lymphocytes in fresh, unstimulated PBMC were now Ag specific. Enzyme-linked immunospot assays confirmed this high frequency of Ag-specific cells, and intracellular IFN-γ staining demonstrated that the majority of these cells produced IFN-γ after peptide stimulation. Moreover, direct ex vivo SIV-specific cytotoxic activity could be detected in PBMC from five of the six DNA/MVA-vaccinated animals, indicating that this epitope-based DNA prime/MVA boost regimen represents a potent method for inducing high levels of functionally active, Ag-specific CD8+ T lymphocytes in non-human primates.

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.

HIV vaccines 1983-2003

Twenty years after the discovery of HIV, there is still no vaccine. This year, an envelope vaccine aimed at stimulating neutralizing antibodies was unable to protect against infection in phase 3 trials. But more than 20 HIV vaccines designed to stimulate T-cell responses are being developed. Will any of them work?

Induction of Multifunctional Human Immunodeficiency Virus Type 1 (HIV-1)-Specific T Cells Capable of Proliferation in Healthy Subjects by Using a Prime-Boost Regimen of DNA- and Modified Vaccinia Virus Ankara-Vectored Vaccines Expressing HIV-1 Gag Coupled to CD8+ T-Cell Epitopes

ABSTRACT A double-blind randomized phase I trial was conducted in human immunodeficiency virus type 1 (HIV-1)-negative subjects receiving vaccines vectored by plasmid DNA and modified vaccinia virus Ankara (MVA) expressing HIV-1 p24/p17 gag linked to a string of CD8+ T-cell epitopes. The trial had two groups. One group received either two doses of MVA.HIVA (2× MVA.HIVA) (n = 8) or two doses of placebo (2× placebo) (n = 4). The second group received 2× pTHr.HIVA followed by one dose of MVA.HIVA (n = 8) or 3× placebo (n = 4). In the pTHr.HIVA-MVA.HIVA group, HIV-1-specific T-cell responses peaked 1 week after MVA.HIVA vaccination in both ex vivo gamma interferon (IFN-γ) ELISPOT (group mean, 210 spot-forming cells/106 cells) and proliferation (group mean stimulation index, 37), with assays detecting positive responses in four out of eight and five out of eight subjects, respectively. No HIV-1-specific T-cell responses were detected in either assay in the 2× MVA.HIVA group or subjects receiving placebo. Using a highly sensitive and reproducible cultured IFN-γ ELISPOT assay, positive responses mainly mediated by CD4+ T cells were detected in eight out of eight vaccinees in the pTHr.HIVA-MVA.HIVA group and four out of eight vaccinees in the 2× MVA.HIVA group. Importantly, no false-positive responses were detected in the eight subjects receiving placebo. Of the 12 responders, 11 developed responses to previously identified immunodominant CD4+ T-cell epitopes, with 6 volunteers having responses to more than one epitope. Five out of 12 responders also developed CD8+ T-cell responses to the epitope string. Induced T cells produced a variety of anti-viral cytokines, including tumor necrosis factor alpha and macrophage inflammatory protein 1β. These data demonstrate that prime-boost vaccination with recombinant DNA and MVA vectors can induce multifunctional HIV-1-specific T cells in the majority of vaccinees.

Design and pre-clinical evaluation of a universal HIV-1 vaccine.

Background One of the big roadblocks in development of HIV-1/AIDS vaccines is the enormous diversity of HIV-1, which could limit the value of any HIV-1 vaccine candidate currently under test. Methodology and Findings To address the HIV-1 variation, we designed a novel T cell immunogen, designated HIVCONSV, by assembling the 14 most conserved regions of the HIV-1 proteome into one chimaeric protein. Each segment is a consensus sequence from one of the four major HIV-1 clades A, B, C and D, which alternate to ensure equal clade coverage. The gene coding for the HIVCONSV protein was inserted into the three most studied vaccine vectors, plasmid DNA, human adenovirus serotype 5 and modified vaccine virus Ankara (MVA), and induced HIV-1-specific T cell responses in mice. We also demonstrated that these conserved regions prime CD8+ and CD4+ T cell to highly conserved epitopes in humans and that these epitopes, although usually subdominant, generate memory T cells in patients during natural HIV-1 infection. Significance Therefore, this vaccine approach provides an attractive and testable alternative for overcoming the HIV-1 variability, while focusing T cell responses on regions of the virus that are less likely to mutate and escape. Furthermore, this approach has merit in the simplicity of design and delivery, requiring only a single immunogen to provide extensive coverage of global HIV-1 population diversity.

Design and construction of an experimental HIV-1 vaccine for a year-2000 clinical trial in Kenya.

Using recent advances in biological and medical sciences, a new candidate human immunodeficiency virus (HIV) vaccine has been developed and tailor-designed for a phase III clinical trial in Kenya. It has two components, DNA and MVA (an attenuated poxvirus), used in a prime-boost vaccination protocol. Both of these vaccine vehicles express a common ‘chimeric’ protein derived from small parts of the HIV genome. The vaccine focuses on the induction of cell-mediated immune responses.

DNA multi-CTL epitope vaccines for HIV and Plasmodium falciparum: immunogenicity in mice

The potential of building multi-cytotoxic T lymphocyte (CTL) epitope antigens in combination with the nucleic acid immunization technology is explored for development of acquired immunodeficiency syndrome (AIDS) and malaria vaccines. A novel minimal vector pTH for direct gene transfer was constructed for efficient expression of vaccine antigens and used as a vehicle for human immunodeficiency virus (HIV)- and Plasmodium falciparum-derived polyepitope genes. Two murine epitopes were included into these constructs to allow for testing of vaccine immunogenicity in small animals. The results showed that a single DNA injection generated CTL responses in all 15 vaccinated mice. The elicited CTL precursor frequencies were estimated in an interferon-gamma (IFN-gamma)-based ELISPOT assay and found to be an average of 300 (range 4-1346) peptide-responding cells per 10(6) splenocytes.

Pre-clinical development of a multi-CTL epitope-based DNA prime MVA boost vaccine for AIDS.

Reliable and effective methods for induction of cytotoxic T-lymphocytes (CTL) are constantly persued. Central to this search is work in animal models, which allow to test novel vaccine strategies and ultimately lead to a more efficient planning of clinical trials. Here, human immunodeficiency virus (HIV) vaccine candidates were constructed as a string of partially overlapping CTL epitopes (20 human, 3 macaque and 1 mouse) delivered and expressed using plasmid DNA and modified virus Ankara (MVA; an attenuated vaccinia virus), which are both vaccine vehicles acceptable for use in humans. In mice, these vaccines were shown to induce virus-specific interferon-gamma-producing and cytolytic CD8+ T-cells after a single intramuscular needle injection. When immunization protocols were sought which would improve the level of induced HIV-specific T-cells, DNA priming-MVA boosting was found to be the most potent protocol. The multi-epitope DNA also elicited CTL when delivered intradermally using the Accell gene delivery device (gene gun). Finally, a combined intradermal gene gun DNA-MVA vaccination regimen induced in macaques high frequencies of circulating CTL, which were comparable to those observed in simian immunodeficiency virus (SIV)-infected monkeys. Further optimization of this method in non-human primates is under way. Thus, a vaccination regimen for an effective elicitation of CTL has been developed which might facilitate evaluation of the role(s) that these lymphocytes play in the control of SIV and HIV infections.

Vaccine-elicited Human T Cells Recognizing Conserved Protein Regions Inhibit HIV-1

Virus diversity and escape from immune responses are the biggest challenges to the development of an effective vaccine against HIV-1. We hypothesized that T-cell vaccines targeting the most conserved regions of the HIV-1 proteome, which are common to most variants and bear fitness costs when mutated, will generate effectors that efficiently recognize and kill virus-infected cells early enough after transmission to potentially impact on HIV-1 replication and will do so more efficiently than whole protein-based T-cell vaccines. Here, we describe the first-ever administration of conserved immunogen vaccines vectored using prime-boost regimens of DNA, simian adenovirus and modified vaccinia virus Ankara to uninfected UK volunteers. The vaccine induced high levels of effector T cells that recognized virus-infected autologous CD4(+) cells and inhibited HIV-1 replication by up to 5.79 log10. The virus inhibition was mediated by both Gag- and Pol- specific effector CD8(+) T cells targeting epitopes that are typically subdominant in natural infection. These results provide proof of concept for using a vaccine to target T cells at conserved epitopes, showing that these T cells can control HIV-1 replication in vitro.

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.