A novel poxvirus-based vaccine, MVA-CHIKV, is highly immunogenic and protects mice against chikungunya infection.

UNLABELLED: 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-ß), 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.

Kinetic and phenotypic analysis of CD8+ T cell responses after priming with alphavirus replicons and homologous or heterologous booster immunizations.

UNLABELLED: Alphavirus replicons are potent inducers of CD8(+) T cell responses and thus constitute an attractive vaccine vector platform for developing novel vaccines. However, the kinetics and memory phenotype of CD8(+) T cell responses induced by alphavirus replicons are not well characterized. Furthermore, little is known how priming with alphavirus replicons affects booster immune responses induced by other vaccine modalities. We demonstrate here that a single immunization with an alphavirus replicon, administered as viral particles or naked DNA, induced an antigen-specific CD8(+) T cell response that had a sharp peak, followed by a rapid contraction. Administering a homologous boost before contraction had occurred did not further increase the response. In contrast, boosting after contraction when CD8(+) T cells had obtained a memory phenotype (based on CD127/CD62L expression), resulted in maintenance of CD8(+) T cells with a high recall capacity (based on CD27/CD43 expression). Increasing the dose of replicon particles promoted T effector memory (Tem) and inhibited T central memory development. Moreover, infection with a replicating alphavirus induced a similar distribution of CD8(+) T cells as the replicon vector. Lastly, the distribution of T cell subpopulations induced by a DNA-launched alphavirus replicon could be altered by heterologous boosts. For instance, boosting with a poxvirus vector (MVA) favored expansion of the Tem compartment. In summary, we have characterized the antigen-specific CD8(+) T cell response induced by alphavirus replicon vectors and demonstrated how it can be altered by homologous and heterologous boost immunizations. IMPORTANCE: Alphavirus replicons are promising vaccine candidates against a number of diseases and are by themselves developed as vaccines against, for example, Chikungunya virus infection. Replicons are also considered to be used for priming, followed by booster immunization using different vaccine modalities. In order to rationally design prime-boost immunization schedules with these vectors, characterization of the magnitude and phenotype of CD8(+) T cell responses induced by alphavirus replicons is needed. Here, we demonstrate how factors such as timing and dose affect the phenotypes of memory T cell populations induced by immunization with alphavirus replicons. These findings are important for designing future clinical trials with alphaviruses, since they can be used to tailor vaccination regimens in order to induce a CD8(+) T cell response that is optimal for control and/or clearance of a specific pathogen.

Prime-boost immunization strategies against Chikungunya virus.

UNLABELLED: 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.

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

UNLABELLED: 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.

Cellular immunogenicity of novel gene immunogens in mice monitored by in vivo imaging.

The efficient cell-mediated immune response clears cells expressing deoxyribonucleic acid (DNA) immunogens, but there are no methods to monitor this in vivo. We hypothesized that immune-mediated clearance can be monitored in vivo if DNA immunogens are coexpressed with reporter(s). To test this, we designed genes encoding human immunodeficiency virus 1 (HIV-1) reverse transcriptase (RT) fused via its N- or C-terminus to 30-amino acid-long Gly-Ala-repeat of Epstein-Barr virus nuclear antigen 1 or via the N-terminus to the transport signal of invariant chain/Ii or inserted between the cytoplasmic and luminal domains of lysosome-associated membrane protein I (LAMP). DNA immunogens mixed with luciferase gene were injected into BALB/c mice with subsequent electroporation. Reporter expression seen as luminescence was monitored by in vivo imaging. When luminescence faded, mice were sacrificed, and their splenocytes were stimulated with RT-derived antigens. Fading of luminescence correlated with the RT-specific secretion of interferon-γ and interleukin-2. Both immune and in vivo imaging techniques concordantly demonstrated an enhanced immunogenicity of RT-LAMP and of the N-terminal Gly-Ala-RT fusion genes. In vivo imaging performed as an animal-sparing method to estimate the overall performance of DNA immunogens, predicting it early in the experiment. So far, in vivo imaging cannot be a substitute for conventional immune assays, but it is supplementary to them. Further experiments are needed to identify which arms of cellular immune response in vivo imaging monitors best.

Rational design of HIV vaccines and microbicides: report of the EUROPRISE annual conference 2011.

Europrise is a Network of Excellence supported by the European Commission within the 6th Framework programme from 2007 to 2012. The Network has involved over 50 institutions from 13 European countries together with 3 industrial partners and 6 African countries. The Network encompasses an integrated program of research, training, dissemination and advocacy within the field of HIV vaccines and microbicides. A central and timely theme of the Network is the development of the unique concept of co-usage of vaccines and microbicides. Training of PhD students has been a major task, and some of these post-graduate students have here summarized novel ideas emanating from presentations at the last annual Europrise meeting in Prague. The latest data and ideas concerning HIV vaccine and microbicide studies are included in this review; these studies are so recent that the majority have yet to be published. Data were presented and discussed concerning novel immunisation strategies; microbicides and PrEP (alone and in combination with vaccines); mucosal transmission of HIV/SIV; mucosal vaccination; novel adjuvants; neutralizing antibodies; innate immune responses; HIV/SIV pathogenesis and disease progression; new methods and reagents. These - necessarily overlapping topics - are comprehensively summarised by the Europrise students in the context of other recent exciting data.

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

BACKGROUND: The 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. METHODS: Herein 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). RESULTS: Neither 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. CONCLUSIONS: These 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.

Rational design of HIV vaccine and microbicides: report of the EUROPRISE annual conference.

EUROPRISE is a Network of Excellence sponsored from 2007 to 2011 by the European Commission within the 6th Framework Program. The Network encompasses a wide portfolio of activities ranging from an integrated research program in the field of HIV vaccines and microbicides to training, dissemination and advocacy. The research program covers the whole pipeline of vaccine and microbicide development from discovery to early clinical trials. The Network is composed of 58 partners representing more than 65 institutions from 13 European countries; it also includes three major pharmaceutical companies (GlaxoSmithKline, Novartis and Sanofi-Pasteur) involved in HIV microbicide and vaccine research. The Network displays a dedicated and informative web page: http://www.europrise.org. Finally, a distinguishing trait of EUROPRISE is its PhD School of students from across Europe, a unique example in the world of science aimed at spreading excellence through training. EUROPRISE held its second annual conference in Budapest in November, 2009. The conference had 143 participants and their presentations covered aspects of vaccine and microbicide research, development and discovery. Since training is a major task of the Network, the students of the EUROPRISE PhD program summarized certain presentations and their view of the conference in this paper.

Attenuated and vectored vaccines protect nonhuman primates against Chikungunya virus.

Chikungunya virus (CHIKV) is rapidly spreading across the globe, and millions are infected. Morbidity due to this virus is a serious threat to public health, but at present, there is no vaccine against this debilitating disease. We have recently developed a number of vaccine candidates, and here we have evaluated 3 of them in a nonhuman primate model. A single immunization with an attenuated strain of CHIKV (Δ5nsP3), a homologous prime-boost immunization with a DNA-launched RNA replicon encoding CHIKV envelope proteins (DREP-E), and a DREP-E prime followed by a recombinant modified vaccinia virus Ankara encoding CHIKV capsid and envelope (MVA-CE) boost all induced protection against WT CHIKV infection. The attenuated Δ5nsP3 virus proved to be safe and did not show any clinical signs typically associated with WT CHIKV infections such as fever, skin rash, lymphopenia, or joint swelling. These vaccines are based on an East/Central/South African strain of Indian Ocean lineage, but they also generated neutralizing antibodies against an isolate of the Asian genotype that now is rapidly spreading across the Americas. These results form the basis for clinical development of an efficacious CHIKV vaccine that generates both humoral and cellular immunity with long-term immunological memory.

Therapeutics and vaccines against chikungunya virus.

Currently, there are no licensed vaccines or therapies available against chikungunya virus (CHIKV), and these were subjects discussed during a CHIKV meeting recently organized in Langkawi, Malaysia. In this review, we chart the approaches taken in both areas. Because of a sharp increase in new data in these fields, the present paper is complementary to previous reviews by Weaver et al. in 2012 and Kaur and Chu in 2013 . The most promising antivirals so far discovered are reviewed, with a special focus on the virus-encoded replication proteins as potential targets. Within the vaccines in development, our review emphasizes the various strategies in parallel development that are unique in the vaccine field against a single disease.

Oral delivery of plant-derived HIV-1 p24 antigen in low doses shows a superior priming effect in mice compared to high doses.

During early infection with human immunodeficiency virus type 1 (HIV-1), there is a rapid depletion of CD4(+) T-cells in the gut-associated lymphoid tissue (GALT) in the gastrointestinal tract. Therefore, immediate protection at these surfaces is of high priority for the development of an HIV-1 vaccine. Thus, transgenic plants expressing HIV-1 antigens, which are exposed to immune competent cells in the GALT during oral administration, can be interesting as potential vaccine candidates. In the present study, we used two HIV-1 p24 antigen-expressing transgenic plant systems, Arabidopsis thaliana and Daucus carota, in oral immunization experiments. Both transgenic plant systems showed a priming effect in mice and induced humoral immune responses, which could be detected as anti-p24-specific IgG in sera after an intramuscular p24 protein boost. Dose-dependent antigen analyses using transgenic A. thaliana indicated that low p24 antigen doses were superior to high p24 antigen doses.

Micro-minicircle Gene Therapy: Implications of Size on Fermentation, Complexation, Shearing Resistance, and Expression.

The minicircle (MC), composed of eukaryotic sequences only, is an interesting approach to increase the safety and efficiency of plasmid-based vectors for gene therapy. In this paper, we investigate micro-MC (miMC) vectors encoding small regulatory RNA. We use a construct encoding a splice-correcting U7 small nuclear RNA, which results in a vector of 650 base pairs (bp), as compared to a conventional 3600 bp plasmid carrying the same expression cassette. Furthermore, we construct miMCs of varying sizes carrying different number of these cassettes. This allows us to evaluate how size influences production, super-coiling, stability and efficiency of the vector. We characterize coiling morphology by atomic force microscopy and measure the resistance to shearing forces caused by an injector device, the Biojector. We compare the behavior of miMCs and plasmids in vitro using lipofection and electroporation, as well as in vivo in mice. We here show that when the size of the miMC is reduced, the formation of dimers and trimers increases. There seems to be a lower size limit for efficient expression. We demonstrate that miMCs are more robust than plasmids when exposed to shearing forces, and that they show extended expression in vivo.Molecular Therapy-Nucleic Acids (2014); doi:10.1038/mtna.2013.67.

Broad and potent cellular and humoral immune responses after a second late HIV-modified vaccinia virus ankara vaccination in HIV-DNA-primed and HIV-modified vaccinia virus Ankara-boosted Swedish vaccinees.

We have previously shown that an HIV vaccine regimen including three HIV-DNA immunizations and a single HIV-modified vaccinia virus Ankara (MVA) boost was safe and highly immunogenic in Swedish volunteers. A median 38 months after the first HIV-MVA vaccination, 24 volunteers received 10(8) plaque-forming units of HIV-MVA. The vaccine was well tolerated. Two weeks after this HIV-MVA vaccination, 18 (82%) of 22 evaluable vaccinees were interferon (IFN)-γ enzyme-linked immunospot (ELISpot) reactive: 18 to Gag and 10 (45%) to Env. A median minimal epitope count of 4 to Gag or Env was found in a subset of 10 vaccinees. Intracellular cytokine staining revealed CD4(+) and/or CD8(+) T cell responses in 23 (95%) of 24 vaccinees, 19 to Gag and 19 to Env. The frequency of HIV-specific CD4(+) and CD8(+) T cell responses was equally high (75%). A high proportion of CD4(+) and CD8(+) T cell responses to Gag was polyfunctional with production of three or more cytokines (40% and 60%, respectively). Of the Env-specific CD4(+) T cells 40% were polyfunctional. Strong lymphoproliferative responses to Aldrithiol-2 (AT-2)-treated subtype A, B, C, and A_E virus were demonstrable in 21 (95%) of 22 vaccinees. All vaccinees developed binding antibodies to Env and Gag. Neutralizing antibodies were detected in a peripheral blood mononuclear cell (PBMC)-based assay against subtype B and CRF01_AE viruses. The neutralizing antibody response rates were influenced by the vaccine dose and/or mode of delivery used at the previous HIV-MVA vaccination. Thus, a second late HIV-MVA boost induced strong and broad cellular immune responses and improved antibody responses. The data support further exploration of this vaccine concept.

A truncated plasmid-encoded HIV-1 reverse transcriptase displays strong immunogenicity.

Besides being an important target in the antiretroviral therapy against the human immunodeficiency virus type 1 (HIV-1), the HIV-1 reverse transcriptase (RT) enzyme has potential as a vaccine antigen. In this study, we explored the ability of plasmid-encoded RT to induce cell-mediated immune responses. The strategy for increasing the immunogenicity of the protein was to delete non- or low-immunogenic parts in order to focus the immune responses to known immunogenic regions. Expression and immunogenicity of the truncated RT was compared to a clinically evaluated full-length RT construct, and the truncated RT displayed enhanced in vitro expression and cell-mediated immune responses in BALB/c and HLA-A0201 transgenic C57BL/6 mice. The strong immune responses were retained also when the truncated RT was delivered as a part of a multigene HIV-1 vaccine. Linking the RT gene to a highly expressed HIV-1 protease gene did not increase the immunogenicity of RT. This optimization strategy could be used to enhance the immunogenicity of other RT-encoding DNA vaccines.

Consensus HIV-1 FSU-A integrase gene variants electroporated into mice induce polyfunctional antigen-specific CD4+ and CD8+ T cells.

Our objective is to create gene immunogens targeted against drug-resistant HIV-1, focusing on HIV-1 enzymes as critical components in viral replication and drug resistance. Consensus-based gene vaccines are specifically fit for variable pathogens such as HIV-1 and have many advantages over viral genes and their expression-optimized variants. With this in mind, we designed the consensus integrase (IN) of the HIV-1 clade A strain predominant in the territory of the former Soviet Union and its inactivated derivative with and without mutations conferring resistance to elvitegravir. Humanized IN gene was synthesized; and inactivated derivatives (with 64D in the active site mutated to V) with and without elvitegravir-resistance mutations were generated by site-mutagenesis. Activity tests of IN variants expressed in E coli showed the consensus IN to be active, while both D64V-variants were devoid of specific activities. IN genes cloned in the DNA-immunization vector pVax1 (pVaxIN plasmids) were highly expressed in human and murine cell lines (>0.7 ng/cell). Injection of BALB/c mice with pVaxIN plasmids followed by electroporation generated potent IFN-γ and IL-2 responses registered in PBMC by day 15 and in splenocytes by day 23 after immunization. Multiparametric FACS demonstrated that CD8+ and CD4+ T cells of gene-immunized mice stimulated with IN-derived peptides secreted IFN-γ, IL-2, and TNF-α. The multi-cytokine responses of CD8+ and CD4+ T-cells correlated with the loss of in vivo activity of the luciferase reporter gene co-delivered with pVaxIN plasmids. This indicated the capacity of IN-specific CD4+ and CD8+ T-cells to clear IN/reporter co-expressing cells from the injection sites. Thus, the synthetic HIV-1 clade A integrase genes acted as potent immunogens generating polyfunctional Th1-type CD4+ and CD8+ T cells. Generation of such response is highly desirable for an effective HIV-1 vaccine as it offers a possibility to attack virus-infected cells via both MHC class I and II pathways.

Immunization with multiple vaccine modalities induce strong HIV-specific cellular and humoral immune responses.

Heterologous priming and boosting with antigens expressed by DNA, viral vectors, or as proteins, are experimental strategies to induce strong immune responses against infectious diseases and cancer. In a preclinical study we compared the ability of recombinant modified vaccinia Ankara encoding HIV antigens (MVA-CMDR), and/or recombinant gp140C (rgp140C), to boost responses induced by a multigene/multisubtype HIV DNA vaccine delivered by electroporation (EP). Homologous DNA immunizations augmented by EP stimulated strong cellular immune responses. Still stronger cellular immune responses were observed after DNA priming and MVA-CMDR boosting, which was superior to all other immunization schedules tested in terms of antigen-specific IFN-γ, IL-2, and bifunctional IFN-γ and IL-2 responses. For HIV Env-specific antibody responses, mice receiving repeated rgp140C immunizations, and mice boosted with rgp140C, elicited the highest binding titers and the highest numbers of antibody-secreting B cells. When considering both cellular and humoral immune responses, a combination of DNA, MVA-CMDR, and rgp140C immunizations induced the overall most potent immune responses and the highest avidity of HIV Env-specific antibodies. These data emphasize the importance of including multiple vaccine modalities that can stimulate both T and B cells, and thus elicit strong and balanced immune responses. The present HIV vaccine combination holds promise for further evaluation in clinical trials.

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

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.

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.

Induction of HIV-1-specific cellular and humoral immune responses following immunization with HIV-DNA adjuvanted with activated apoptotic lymphocytes.

Delivery of DNA encoding foreign antigens into mammalian cells can induce adaptive immune responses. There are currently many DNA-based vaccines in clinical trials against infectious diseases and cancer but there is a lack of adjuvants for improvement of responses to DNA-based vaccines. Here, we show augmented systemic and mucosa-associated B cell responses after immunization with a cocktail of seven different plasmids (3 env, 2 gag, 1 rev, 1 RT) combined with mitogen activated apoptotic syngeneic lymphocytes in mice. In addition we show that apoptotic cells can function as adjuvant for induction of cellular immune responses in a magnitude comparable to the cytokine adjuvant GM-CSF in mice. These data suggest that activated apoptotic lymphocytes can act independent as adjuvants to improve antigen-specific DNA vaccines.