Membrane and envelope virus proteins co-expressed as lysosome associated membrane protein (LAMP) fused antigens: a potential tool to develop DNA vaccines against flaviviruses

Vaccination is the most practical and cost-effective strategy to prevent the majority of the flavivirus infection to which there is an available vaccine. However, vaccines based on attenuated virus can potentially promote collateral side effects and even rare fatal reactions. Given this scenario, the development of alternative vaccination strategies such as DNA-based vaccines encoding specific flavivirus sequences are being considered. Endogenous cytoplasmic antigens, characteristically plasmid DNA-vaccine encoded, are mainly presented to the immune system through Major Histocompatibility Complex class I – MHC I molecules. The MHC I presentation via is mostly associated with a cellular cytotoxic response and often do not elicit a satisfactory humoral response. One of the main strategies to target DNA-encoded antigens to the MHC II compartment is expressing the antigen within the Lysosome-Associated Membrane Protein (LAMP). The flavivirus envelope protein is recognized as the major virus surface protein and the main target for neutralizing antibodies. Different groups have demonstrated that co-expression of flavivirus membrane and envelope proteins in mammalian cells, fused with the carboxyl-terminal of LAMP, is able to induce satisfactory levels of neutralizing antibodies. Here we reviewed the use of the envelope flavivirus protein co-expression strategy as LAMP chimeras with the aim of developing DNA vaccines for dengue, West Nile and yellow fever viruses.

while vaccines for dengue and West Nile remain elusive goals.The main strategy for flavivirus vaccine development has been the attenuation of specific strains, such as the YF 17D and the Japanese encephalitis SA14-14-2, or virus inactivation as the tick-borne encephalitis.However, the use of attenuated virus strains can potentially promote harmful effects, such as increased symptom severity and seldom fatal reactions (Liu 2003).In addition, such vaccines are not recommended for in-fants less than 9 months old, pregnant women and immunodeficient subjects (Cetron et al. 2002).
The first human flavivirus-attenuated vaccine was the vaccine against YF (17D).In the last 70 years, more than 400 million people worldwide have been vaccinated with the 17D vaccine with a remarkable record of safety and efficacy (Putnak et al. 2003).The 17D vaccine generates both long-lasting neutralizing antibodies and a T cell response (Poland et al. 1981, Reinhardt et al. 1998).However, despite several improvements made in the manufacture and quality control process, increased severity of symptoms (Monath et al. 2002) and fatal reactions (Vasconcelos et al. 2001, Lefeuvre et al. 2004) has been systematically associated with the YF virus-attenuated vaccination.Given this scenario, the development of alternative vaccination strategies, such as DNA-based vaccines encoding specific flavivirus sequences, has been considered (Donnelly et al. 1997, Lewis and Babiuk 1999, Robinson 1999, Schultz et al. 2000) and presents some advantages (Table I).

LYSOSOME-ASSOCIATED MEMBRANE PROTEIN (LAMP) TARGET STRATEGY
Endogenous cytoplasmic antigens, typical of DNA encoded antigens, are mainly presented to the immune system through the Major Histocompatibility Complex (MHC) class I molecules, which are mostly associated with cellular cytotoxic response.However, such responses do not elicit a satisfactory humoral response very often, which is essential for efficient virus neutralization.In fact, the activation of CD4 + T cells is important to support CD8 + T cell responses and the development of memory, antibody class switching and clonal expansion of antigen-specific B cells (Rocha and Tanchot 2004).The activation of CD4 + T cells requires the Antigen-Presenting Cells (APCs) with antigenic peptides loaded into the groove of MHC class II molecules.The loading of the antigenic peptide takes place in intracellular organelles rich in MCH class II molecules, termed MIIC (Kleijmeer et al. 1997, Drake et al. 1999).Lysosome-associated membrane protein (LAMP) molecules, which are naturally found in the outer membrane of lysosomes (Lippincott-Schwartz and Fambrough 1986), traffic through the MIIC and the in-frame expression of antigens within LAMP, in plasmid DNA constructs, is able to drive the new translated chimerical antigen into the MIIC.LAMP/antigen chimeras, like LAMP/HIV Gag (Marques et al. 2003, Chikhlikar et al. 2004, Arruda et al. 2004, 2006) and LAMP/dengue virus 2 (Raviprakash et al. 2000, Lu et al. 2003), have been shown to target the antigens to MIIC and were found to elicit enhanced immune responses when compared to DNA vaccines encoding unmodified native antigens.
Considering that the flavivirus envelope (E) protein is recognized as the major virus surface protein and the main target for neutralizing antibodies, this antigen constitutes a potential target for DNA vaccines development initiatives.The targeting of flavivirus E protein to the MIIC has been shown to enhance neutralizing antibody (Turley et al. 2000) production in immunized mice (Raviprakash et al. 2001, Donnelly et al. 2003, Anwar et al. 2005) and in non-human primates (Dr.Kanakatte Raviprakash, personal communication).

DEVELOPMENT OF DNA-BASED VACCINES AGAINST FLAVIVIRUSES: MEMBRANE-ENVELOPE PROTEINS CO-EXPRESSED AS LAMP FUSED ANTIGENS
The genome of the yellow fever virus, the prototype of the flavivirus family, is organized in a single-stranded positive-sense RNA molecule of ∼10.8Kb, flanked by a 5' cap and a 3' non-polyadenylated terminal loop structure.It encodes three genes for structural proteins (Capsid, Membrane -M, and E) and seven genes for nonstructural (NS) proteins (NS1, NS2a, NS2b, NS3, NS4a, NS4b, and NS5).Co-expression of flavivirus M and E genes in mammalian cells has shown to produce Virus-Like Particles (VLPs) containing M and E proteins (Raviprakash et al. 2000, Lu et al. 2003, Wu et al. 2006).The VLP structure preserves viral conformational epitopes that are responsible to induce B-cells neutralizing antibodies production and, as a consequence, virus infection neutralization (Wu et al. 2006).Different groups previously described the development of flavivirus DNA vaccines based on the expression of different viruses' M and E regions as wild type proteins, as well as chimerical proteins associated to LAMP.The M and E flavivirus proteins co-expressed as LAMP fused antigens, delivered as DNA vaccines, seems to be a promising approach to prevent flavivirus infections.Our research group is currently using this technology aim- ing to develop DNA vaccines against West Nile, dengue and yellow fever viruses.All wild type DNA constructs comprise a region extending from the capsid reticulum endoplasmic (ER) translocation signal to the E protein transmembrane domain.In parallel, the fused LAMP constructs were obtained through the replacement of E protein transmembrane domain by the C-terminal do-main of LAMP (Fig. 1).Despite the substitution of the E protein transmembrane domain by LAMP, M-E-LAMP constructs also seem to be able to generate VLPs (Raviprakash et al. 2001).Raviprakash and co-workers ( 2001) first evaluated M and E co-expression strategy as wild type and LAMPfused proteins, in terms of localization and immuno- Lu and co-workers ( 2003) continued the studies on M and E co-expression to evaluate the memory response.First, the authors evaluated the expression of both dengue proteins by Western blot analysis, confirming the expected specific bands corresponding to the glycosylated and processed protein forms of both the native E and the E/LAMP chimera.The lysosomal-like distribution of M-E/LAMP and the co-localization of MHC II and LAMP molecules were also confirmed through fluorescence and confocal microscopy.To evaluate the immunogenicity of their vaccines, M-E and M-E/LAMP encoded DNA constructs, the immune responses of the immunized mice were followed for 1 year.Both DNA constructs were used for mice immunization and the antibody response was measured by Enzyme Linked Immuno Sorbent Assay -ELISA.None of the serum samples of the M-E DNA immunized group showed a significant titer of virus neutralization, while sera of the M-E/LAMP DNA immunized group showed strong virus neutralization capability.Anwar and co-workers (2005) used the M-E/LAMP strategy to develop a West Nile (WN) DNA vaccine.Their Western blot findings showed an increased intracellular concentration of the M-E/LAMP chimerical protein in transfected cells when compared to the native protein.This result pointed to a natural cellular secretion of E protein that may be retarded by the association between the LAMP signal and the outer membrane of the lysosome.The confocal and immunoelectron microscopy analyses showed the typical lysosomal distribution of the WN M-E/LAMP and the co-localization with endogenous LAMP, MHC class II, and H-2M molecules, the latter of which is closely related to antigen presentation.To evaluate the immunogenicity of the WN vaccines, M-E and M-E/LAMP DNAs were injected in mice that were followed for 2 years after the immunization.Mice immunized with M-E showed appreciable endpoint titers, but their antibody responses were not sustained after 3 months.In contrast, mice immunized with M-E/LAMP showed high antibody responses, with increasing titers from days 55 to 125, and a significant neutralization titer response at days 90 and 125.To assess the presence of memory B cell response, the mice were boosted 19 months after the last immunization, and the blood collected 2 weeks later.While there was no significant neutralization activity in the sera from the M-E DNA immunized group, the sera from the M-E/LAMP DNA immunized group showed significant neutralization titers.
We are currently evaluating a DNA construct expressing the envelope of the YF, fused to LAMP molecules, in murine immunization experiments.Immunizations with YF M-E/LAMP encoded DNA showed high titers of neutralizing antibodies that were able to protect the animals from the intracerebral challenge with the YF virus.In addition, when compared to the attenuated 17DD YF virus vaccine, the plasmid DNA expression of the E protein within LAMP induced similar CD4 + and CD8 + T cell responses, qualitatively and quantitatively (data not shown).

FUTURE DIRECTIONS
In order to improve the DNA-codified antigen expression in human cells, we are currently developing codonoptimized vaccines by using a genetic algorithm (LETO 1.0 -Entelechon ).Parameters as codon usage, message RNA secondary structures, cryptic splice sites, and internal restriction sites, among others, have been modified and/or removed from the native sequences.The blend of LAMP fusion and codon optimization technologies shows promising potential and may lead to new and better DNA vaccines.Our preliminary evaluations of the novel YF optimized DNA vaccine constructs address the levels and localization of the protein expression.By immunofluorescence assays, the translation efficiency of the optimized vaccines was considered at least 20 times higher (data not shown).Considering the higher efficiency of the optimized M-E/LAMP DNA constructs (M-E OPT /LAMP), compared to the nonoptimized version, we believe that the optimized version will be able to induce much stronger T and B cell responses in animal models.Ultimately, the improvement of the level of antigen expression will allow us to reduce the number and concentration of the DNA vaccine dose used in in vivo immunization experiments.

Fig. 1 -
Fig. 1 -Membrane-Envelope flavivirus DNA vaccine constructs: co-expression as wild type and as LAMP fused antigens.Polymerase Chain Reaction -PCR amplification was performed to obtain Membrane (M) and Envelope (E) co-expressed flavivirus-based DNA vaccines.To generate the M-E wild type constructs, specific primers were designed to amplify a fragment extending from the capsid reticulum endoplasmic (ER) translocation signal to the E protein transmembrane domain (cap-pM/M-Env or M-E).To generate the M-E/LAMP fused constructs, specific reverse primers were designed to annealed just before the envelope transmembrane domain to allow its substitution by the Carboxi-terminal domain of the Lysosome Associated Membrane Protein -LAMP (Cap-pM/M-Env-LAMP or M-E/LAMP).

TABLE I Comparison between virus-attenuated and wild type and LAMP fused DNA-based vaccines.
An Acad BrasCienc (2009) 81 (4) ET AL. 2003.HIV-1 p55Gag encoded in the lysosome-associated membrane protein-1 as a DNA plasmid vaccine chimera is highly expressed, traffics to the major histocompatibility class II compartment, and elicits enhanced immune responses.J Biol Chem 278: 37926-37936.MONATH TP ET AL. 2002.Single mutation in the flavivirus envelope protein hinge region increases neurovirulence for mice and monkeys but decreases viscerotropism for monkeys: relevance to development and safety testing of live, attenuated vaccines.J Virol 76: 1932-1943.POLAND JD ET AL. 1981.Persistence of neutralizing antibody 30-35 years after immunization with 17D yellow fever vaccine.Bull World Health Organ 59: 895-900.PUTNAK R ET AL. 2003.DNA vaccines for flaviviruses.Adv Virus Res 61: 445-468.RAVIPRAKASH K ET AL. 2000.Immunogenicity of dengue virus type 1 DNA vaccines expressing truncated and full length envelope protein.Vaccine 18: 2426-2434.