Strategies to enhance the potency of HIV-1 DNA vaccines

Despite 30 years of intense research on HIV/AIDS, we have yet to arrive at a prophylactic vaccine that confers complete protection. This mission is complicated by the virus’s vast genetic variability and its ability to mask the targets for neutralizing antibodies. In addition, HIV infects a subset of immune cells that normally coordinate the immune system and integrates its genome into the DNA of the infected cell. The failure of early HIV vaccine candidates based on classical vaccine strategies has underscored the importance of exploring alternative vaccine approaches, including DNA vaccines. These vaccines are capable of inducing broad cell-mediated and humoral immune responses and their potential is indicated by the licensing of DNA vaccines for veterinary use and by the induction of protection against infectious diseases in animal models. Still, further efforts are needed to ultimately make this approach efficacious in humans. This thesis describes means of enhancing the potency of DNA vaccines for HIV-1, such as by optimization of the gene insert, use of delivery devices and combinations of vaccine candidates. In one project, we constructed DNA vaccines expressing different variants of the HIV-1 protease and determined that both in vitro expression and immunogenicity of the encoded protein in mice were drastically enhanced when a point mutation was introduced in the active site of the protease enzyme, rendering it inactive. We thus discovered a means of enhancing the immunogenicity of HIV-1 protease. Another project was designed to establish an immunization protocol for electroporation (EP)-mediated intradermal DNA vaccine delivery. We showed that a straightforward protocol, using repeated intradermal EP immunizations with a rather short immunization interval, induced strong and long-lived immune responses. A novel FluoroSpot assay detecting vaccine-specific secretion of gamma interferon (IFN-γ) and/or interleukin-2 (IL-2) was shown to possess the advantages of both ELISpot and intracellular staining. Further evidence supporting the use of EP for the delivery of DNA vaccines was obtained in a study where a combination of jet injection and EP, but not needle plus EP or jet injection alone, was able to overcome dose restrictions of DNA vaccination and induce higher antibody and cytotoxic T cell responses when the DNA dose was increased to a considerably higher level. This shows that two optimized DNA vaccine delivery devices can act together to overcome dose limitations of a plasmid DNA vaccine. Experiments evaluating the combined effect of different vaccine modalities were conducted. In one study, two plasmids included in the clinically evaluated HIVIS multigene/multisubtype HIV vaccine encoding Env and Rev were combined with the Auxo-GTU-MultiHIV multigene DNA vaccine that is primarily designed to induce cell-mediated immune responses. Immunization of mice revealed that strong immune responses against the two vaccine modalities were retained, with only a slight reduction of cellular immune responses when the vaccines were administered to the same mice. Moreover, heterologus prime-boost immunizations of mice with DNA, recombinant vaccinia vector (MVA-CMDR) and recombinant protein (rgp140C) induced potent cell-mediated and humoral immune responses and demonstrated the importance of including DNA priming immunizations. These attempts to enhance the potency of DNA vaccines will potentially contribute to the understanding of how to construct, deliver and compose the next generation of DNA vaccines against HIV as well as other infectious diseases and cancers

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

Improving the immunogenicity of HIV-1 DNA vaccines.

Nearly 30 years have passed since the start of the global HIV epidemic and we are still unable to control the spread of the virus. HIV predominately infects cells crucial for the function of the immune system, integrates into the host genome and demonstrates a huge genetic variability, and the use of therapeutic antiretroviral drugs can restrain but not clear the infection. Therefore a protective vaccine is considered the best approach to counteract HIV. The failure of HIV vaccine candidates based on classical vaccine strategies have paved the way for novel vaccine modalities, such as genetic vaccines. These vaccines have induced broad and robust immune responses in animals but need to be optimized to ultimately induce protection against HIV infection. Furthermore, no definite correlates of protection against HIV infection are yet identified and this severely complicates the vaccine development. Nevertheless, the licensure of several DNA vaccines for veterinary use and the induction of protection against SIV infection/disease in non‐human primate models raise hope for the possibility to induce protection against HIV‐1/AIDS by DNA vaccination also in humans. This thesis describes both the effect of optimizing the gene insert and the use of adjuvants to augment the immune responses after immunization with HIV‐1 plasmids. A HIV‐1 protease gene was genetically optimized by changing the amino acid composition. By altering the enzymatic active site, rendering the protein inactive, it was possible to greatly increase the in vitro protein expression and significantly increase the immunogenicity of the gene in various mouse strains, including mice transgenic for the human HLA‐A0201 molecule. We thus identified a rather simple strategy to drastically increase the immunogenicity of HIV‐1 protease and induce strong immune responses against wild type protease as well as against protease carrying drug resistance mutations. The optimized protease construct will be integrated into the clinically evaluated multigene vaccine, HIVIS, and initial results have shown that the immunogenicity of the protease construct, as well as the immunogenicity of the other constructs in the vaccine, is not negatively affected by the addition of the new plasmid. Also, by administering our multigene vaccine formulated in a lipid‐based adjuvant intranasally to young mice we could increase both the systemic cellular and humoral immune responses. In addition, antibody responses could be detected at mucosal sites distant from the intranasal mucosa, demonstrating the ability of DNA vaccines to induce broad immune responses in different compartments of the body. The induction of these mucosal anti‐HIV antibodies presents a possible means to prevent infection at the mucosal surface where the majority of HIV transmissions take place. These and other ways to augment the induction of strong immune responses by DNA vaccination will hopefully provide clues on how to construct and deliver the next generation of potent DNA vaccines against HIV as well as against other infectious diseases and cancers

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

Background The gut associated lymphoid tissue (GALT) includes around two thirds of the total lymphoid system. CD4+ T-cells in the GALT are a main target for HIV during primary infection. Thus, immunization targetting GALT is likely to be of importance for an effective vaccine strategy. Transgenic plants expressing HIV antigens can reach GALT conveniently. This system allows multiple boosts, has simple logistics (no cold chain, no injections) and large production capacity. Methods Three groups of mice were given extract from plant lines expressing HIV-1 p24 at (A) low level (20 ng/feeding); (B) high level (460 ng/feeding); (C) control (wild type, 0 ng). No adjuvant was included. The extracts were administered by gastric tube day 0, 14 and 28. On day 55 all mice were given an intramuscular (i.m.) boost with 10 micrograms of purified p24 antigen. Immune responses were determined by measurement of p24-antibodies in serum by ELISA. Results The mice immunized by the low dose plant line (A) showed a higher systemic immune response after i.m. boost compared to the high dose group (B). The w.t. controls (C) had undetectable p24-responses. The responses in group A were 3 to 10 times higher (ELISA OD values) than in group B. Pre-boost antibody responses were at background levels in all groups. Preliminary analyses indicate a predomninant Th1-type response (antigen-specific IgG2a higher than IgG1). Conclusion Simple and inexpensive means of vaccination are important in order to reach large numbers of people with effective vaccine regimens. The HIV-1 p24 low dose transgenic plant extracts given orally showed a superior priming effect in mice compared to the p24 high dose extracts. This could be an immunization method and route worth exploring further.Molekylärt jordbrukMolekylär biokemiUtveckling av slemhinneadministrerade vaccine

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

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

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

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 pathway

Therapeutics and Vaccines Against Chikungunya Virus

Erratum in Correction to: Vector Borne Zoonotic Dis 2015;15(4):250-257 DOI: 10.1089/vbz.2014.1681. [Vector Borne Zoonotic Dis. 2015]International audienceCurrently, 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