David Hallengärd

A Novel Poxvirus-based Vaccine (MVA-CHIKV) is Highly Immunogenic and Protects Mice against Chikungunya Infection

ABSTRACT There is a need to develop a single and highly effective vaccine against the emerging chikungunya virus (CHIKV), which causes a severe disease in humans. Here, we have generated and characterized the immunogenicity profile and the efficacy of a novel CHIKV vaccine candidate based on the highly attenuated poxvirus vector modified vaccinia virus Ankara (MVA) expressing the CHIKV C, E3, E2, 6K, and E1 structural genes (termed MVA-CHIKV). MVA-CHIKV was stable in cell culture, expressed the CHIKV structural proteins, and triggered the cytoplasmic accumulation of Golgi apparatus-derived membranes in infected human cells. Furthermore, MVA-CHIKV elicited robust innate immune responses in human macrophages and monocyte-derived dendritic cells, with production of beta interferon (IFN-β), proinflammatory cytokines, and chemokines. After immunization of C57BL/6 mice with a homologous protocol (MVA-CHIKV/MVA-CHIKV), strong, broad, polyfunctional, and durable CHIKV-specific CD8+ T cell responses were elicited. The CHIKV-specific CD8+ T cells were preferentially directed against E1 and E2 proteins and, to a lesser extent, against C protein. CHIKV-specific CD8+ memory T cells of a mainly effector memory phenotype were also induced. The humoral arm of the immune system was significantly induced, as MVA-CHIKV elicited high titers of neutralizing antibodies against CHIKV. Remarkably, a single dose of MVA-CHIKV protected all mice after a high-dose challenge with CHIKV. In summary, MVA-CHIKV is an effective vaccine against chikungunya virus infection that induced strong, broad, highly polyfunctional, and long-lasting CHIKV-specific CD8+ T cell responses, together with neutralizing antibodies against CHIKV. These results support the consideration of MVA-CHIKV as a potential vaccine candidate against CHIKV. IMPORTANCE We have developed a novel vaccine candidate against chikungunya virus (CHIKV) based on the highly attenuated poxvirus vector modified vaccinia virus Ankara (MVA) expressing the CHIKV C, E3, E2, 6K, and E1 structural genes (termed MVA-CHIKV). Our findings revealed that MVA-CHIKV is a highly effective vaccine against chikungunya virus, with a single dose of the vaccine protecting all mice after a high-dose challenge with CHIKV. Furthermore, MVA-CHIKV is highly immunogenic, inducing strong innate responses: high, broad, polyfunctional, and long-lasting CHIKV-specific CD8+ T cell responses, together with neutralizing antibodies against CHIKV. This work provides a potential vaccine candidate against CHIKV.

Novel attenuated Chikungunya vaccine candidates elicit protective immunity in C57BL/6 mice

ABSTRACT Chikungunya virus (CHIKV) is a reemerging mosquito-borne alphavirus that has caused severe epidemics in Africa and Asia and occasionally in Europe. As of today, there is no licensed vaccine available to prevent CHIKV infection. Here we describe the development and evaluation of novel CHIKV vaccine candidates that were attenuated by deleting a large part of the gene encoding nsP3 or the entire gene encoding 6K and were administered as viral particles or infectious genomes launched by DNA. The resulting attenuated mutants were genetically stable and elicited high magnitudes of binding and neutralizing antibodies as well as strong T cell responses after a single immunization in C57BL/6 mice. Subsequent challenge with a high dose of CHIKV demonstrated that the induced antibody responses protected the animals from viremia and joint swelling. The protective antibody response was long-lived, and a second homologous immunization further enhanced immune responses. In summary, this report demonstrates a straightforward means of constructing stable and efficient attenuated CHIKV vaccine candidates that can be administered either as viral particles or as infectious genomes launched by DNA. IMPORTANCE Similar to other infectious diseases, the best means of preventing CHIKV infection would be by vaccination using an attenuated vaccine platform which preferably raises protective immunity after a single immunization. However, the attenuated CHIKV vaccine candidates developed to date rely on a small number of attenuating point mutations and are at risk of being unstable or even sensitive to reversion. We report here the construction and preclinical evaluation of novel CHIKV vaccine candidates that have been attenuated by introducing large deletions. The resulting mutants proved to be genetically stable, attenuated, highly immunogenic, and able to confer durable immunity after a single immunization. Moreover, these mutants can be administered either as viral particles or as DNA-launched infectious genomes, enabling evaluation of the most feasible vaccine modality for a certain setting. These CHIKV mutants could represent stable and efficient vaccine candidates against CHIKV.

Biodistribution, persistence and lack of integration of a multigene HIV vaccine delivered by needle-free intradermal injection and electroporation.

Abstract It is likely that gene-based vaccines will enter the human vaccine area soon. A few veterinary vaccines employing this concept have already been licensed, and a multitude of clinical trials against infectious diseases or different forms of cancer are ongoing. Highly important when developing novel vaccines are the safety aspects and also new adjuvants and delivery techniques needs to be carefully investigated so that they meet all short- and long-term safety requirements. One novel in vivo delivery method for plasmid vaccines is electroporation, which is the application of short pulses of electric current immediately after, and at the site of, an injection of a genetic vaccine. This method has been shown to significantly augment the transfection efficacy and the subsequent vaccine-specific immune responses. However, the dramatic increase in delivery efficacy offered by electroporation has raised concerns of potential increase in the risk of integration of plasmid DNA into the host genome. Here, we demonstrate the safety and lack of integration after immunization with a high dose of a multigene HIV-1 vaccine delivered intradermally using the needle free device Biojector 2000 together with electroporation using Derma Vax™ DNA Vaccine Skin Delivery System. We demonstrate that plasmids persist in the skin at the site of injection for at least four months after immunization. However, no association between plasmid DNA and genomic DNA could be detected as analyzed by qPCR following field inversion gel electrophoresis separating heavy and light DNA fractions. We will shortly initiate a phase I clinical trial in which healthy volunteers will be immunized with this multiplasmid HIV-1 vaccine using a combination of the delivery methods jet-injection and intradermal electroporation.

Comparison of plasmid vaccine immunization schedules using intradermal in vivo electroporation.

ABSTRACT In vivo electroporation (EP) has proven to significantly increase plasmid transfection efficiency and to augment immune responses after immunization with plasmids. In this study, we attempted to establish an immunization protocol using intradermal (i.d.) EP. BALB/c mice were immunized with a plasmid encoding HIV-1 p37Gag, either i.d. with the Derma Vax EP device, intramuscularly (i.m.) without EP, or with combinations of both. A novel FluoroSpot assay was used to evaluate the vaccine-specific cellular immune responses. The study showed that i.d. EP immunizations induced stronger immune responses than i.m. immunizations using a larger amount of DNA and that repeated i.d. EP immunizations induced stronger immune responses than i.m. priming followed by i.d. EP boosting. Two and three i.d. EP immunizations induced immune responses of similar magnitude, and a short interval between immunizations was superior to a longer interval in terms of the magnitude of cellular immune responses. The FluoroSpot assay allowed for the quantification of vaccine-specific cells secreting either gamma interferon (IFN-γ), interleukin-2 (IL-2), or both, and the sensitivity of the assay was confirmed with IFN-γ and IL-2 enzyme-linked immunosorbent spot (ELISpot) assays. The data obtained in this study can aid in the design of vaccine protocols using i.d. EP, and the results emphasize the advantages of the FluoroSpot assay over traditional ELISpot assay and intracellular staining for the detection and quantification of bifunctional vaccine-specific immune responses.

Prime-Boost Immunization Strategies against Chikungunya Virus

ABSTRACT Chikungunya virus (CHIKV) is a reemerging mosquito-borne alphavirus that causes debilitating arthralgia in humans. Here we describe the development and testing of novel DNA replicon and protein CHIKV vaccine candidates and evaluate their abilities to induce antigen-specific immune responses against CHIKV. We also describe homologous and heterologous prime-boost immunization strategies using novel and previously developed CHIKV vaccine candidates. Immunogenicity and efficacy were studied in a mouse model of CHIKV infection and showed that the DNA replicon and protein antigen were potent vaccine candidates, particularly when used for priming and boosting, respectively. Several prime-boost immunization strategies eliciting unmatched humoral and cellular immune responses were identified. Further characterization by antibody epitope mapping revealed differences in the qualitative immune responses induced by the different vaccine candidates and immunization strategies. Most vaccine modalities resulted in complete protection against wild-type CHIKV infection; however, we did identify circumstances under which certain immunization regimens may lead to enhancement of inflammation upon challenge. These results should help guide the design of CHIKV vaccine studies and will form the basis for further preclinical and clinical evaluation of these vaccine candidates. IMPORTANCE As of today, there is no licensed vaccine to prevent CHIKV infection. In considering potential new vaccine candidates, a vaccine that could raise long-term protective immunity after a single immunization would be preferable. While humoral immunity seems to be central for protection against CHIKV infection, we do not yet fully understand the correlates of protection. Therefore, in the absence of a functional vaccine, there is a need to evaluate a number of different candidates, assessing their merits when they are used either in a single immunization or in a homologous or heterologous prime-boost modality. Here we show that while single immunization with various vaccine candidates results in potent responses, combined approaches significantly enhance responses, suggesting that such approaches need to be considered in the further development of an efficacious CHIKV vaccine.

Late administration of plasmid DNA by intradermal electroporation efficiently boosts DNA-primed T and B cell responses to carcinoembryonic antigen

Heterologous boost immunisation is considered the most efficient way to enhance DNA-primed immune responses. We have previously shown that administration of recombinant carcinoembryonic antigen (CEA) efficiently boosts humoral responses in mice primed with CEA DNA. However, clinical grade recombinant proteins are far more intriguing to produce than plasmid DNA. Therefore, the possibility to use plasmid DNA for both priming and boosting would be beneficial. With the prospect of future use in a clinical trial, we investigated if electroporation-mediated delivery of DNA could be used to boost DNA-primed immune responses to CEA. The Biojector was used to prime BALB/c mice intradermally three times with CEA66 DNA, encoding an intracellular modified form of CEA. Twelve weeks after the last prime, the animals received either one injection of recombinant CEA or one intradermal injection of twtCEA DNA, encoding the wild type CEA fused to a tetanus T helper epitope, in combination with electroporation. Boosting with rCEA protein did not enhance T cell responses to CEA but induced CEA-specific IgG in 4 of 8 mice. In contrast, intradermal delivery of twtCEA DNA by electroporation led to a tenfold increase in IFN-gamma-producing CD8+ T cells, compared to the levels obtained after the third priming immunisation. The DNA boost also induced high CEA-specific IgG titers in all immunised animals (8/8). The data suggests that a late DNA boost, in combination with enhanced DNA delivery by electroporation, could be used to enhance the efficiency of DNA vaccination and substitute for a heterologous protein boost vaccination.

Intranasal immunization of young mice with a multigene HIV-1 vaccine in combination with the N3 adjuvant induces mucosal and systemic immune responses

One of the major challenges for the development of an HIV vaccine is to induce potent virus-specific immune responses at the mucosal surfaces where transmission of virus occurs. Intranasal delivery of classical vaccines has been shown to induce good mucosal antibody responses, but so far for genetic vaccines the success has been limited. This study shows that young individuals are sensitive to nasal immunization with a genetic vaccine delivered in a formulation of a lipid adjuvant, the Eurocine N3. Intranasal delivery of a multiclade/multigene HIV-1 genetic vaccine gave rise to vaginal and rectal IgA responses as well as systemic humoral and cellular responses. As electroporation might become the preferred means of delivering genetic vaccines for systemic HIV immunity, nasal delivery by droplet formulation in a lipid adjuvant might become a means of priming or boosting the mucosal immunity.

A combination of intradermal jet-injection and electroporation overcomes in vivo dose restriction of DNA vaccines

BackgroundThe use of optimized delivery devices has been shown to enhance the potency of DNA vaccines. However, further optimization of DNA vaccine delivery is needed for this vaccine modality to ultimately be efficacious in humans.MethodsHerein we evaluated antigen expression and immunogenicity after intradermal delivery of different doses of DNA vaccines by needle or by the Biojector jet-injection device, with or without the addition of electroporation (EP).ResultsNeither needle injection augmented by EP nor Biojector alone could induce higher magnitudes of immune responses after immunizations with a high dose of DNA. After division of a defined DNA dose into multiple skin sites, the humoral response was particularly enhanced by Biojector while cellular responses were particularly enhanced by EP. Furthermore, a close correlation between in vivo antigen expression and cell-mediated as well as humoral immune responses was observed.ConclusionsThese results show that two optimized DNA vaccine delivery devices can act together to overcome dose restrictions of plasmid DNA vaccines.

Rational design of HIV vaccines and microbicides: report of the EUROPRISE network annual conference 2010

Novel, exciting intervention strategies to prevent infection with HIV have been tested in the past year, and the field is rapidly evolving. EUROPRISE is a network of excellence sponsored by the European Commission and concerned with a wide range of activities including integrated developmental research on HIV vaccines and microbicides from discovery to early clinical trials. A central and timely theme of the network is the development of the unique concept of co-usage of vaccines and microbicides. This review, prepared by the PhD students of the network captures much of the research ongoing between the partners. The network is in its 5th year and involves over 50 institutions from 13 European countries together with 3 industrial partners; GSK, Novartis and Sanofi-Pasteur. EUROPRISE is involved in 31 separate world-wide trials of Vaccines and Microbicides including 6 in African countries (Tanzania, Mozambique, South Africa, Kenya, Malawi, Rwanda), and is directly supporting clinical trials including MABGEL, a gp140-hsp70 conjugate trial and HIVIS, vaccine trials in Europe and Africa.

Increased expression and immunogenicity of HIV-1 protease following inactivation of the enzymatic activity.

HIV-1 protease is an important target for anti-HIV therapy but has not received much attention as a vaccine antigen. To investigate the immunogenic properties of HIV-1 protease, we designed DNA plasmids encoding variants of the protease gene. Mutations resulting in enzymatic inactivation (D25N) and resistance to standard antiretroviral drugs (V82F/I84V) were introduced in order to examine the impact of the enzymatic activity on immunogenicity and the possibility to induce immune responses against drug resistant protease, respectively. The enzymatic inactivation of protease resulted in significantly increased in vitro expression as well as in vivo immunogenicity. The inactivated protease was highly immunogenic in both BALB/c and HLA-A0201 transgenic C57Bl/6 mice, and the immunogenicity was retained when the gene was delivered as a part of a multigene HIV-1 DNA vaccine. The drug resistance mutations hampered both the cellular and humoral immune responses, as the mutations also affect both CD4 and CD8 T cell epitopes. Taken together, our data demonstrates the possibility to drastically increase the immunogenicity of HIV-1 protease.