Production of Recombinant HIV-1 Antigen in Transgenic Tobacco

Source from:  Tuesday, January 09, 2007 By Patricia Obregon 01/10/2007

In 2004 an estimated forty-two million human immunodeficiency virus (HIV) infections were found throughout the world, and more than 95% of the cases and deaths from AIDS occurred in developing countries. The continuing spread of the epidemic and the high rate of infected people in that area of the world have raised the need to establish urgent preventive measures and extend antiretroviral therapy access (HAART). However, an ongoing serious concern is that these crucial measures are only accessible to a minor number of people who need them, and the introduction of HAART has been unable to slow the progress of HIV in these countries. Therefore, the development of an efficient and cost-effective HIV vaccine has become an urgent need, and the advance of new strategies will be necessary to halt the spread of HIV/AIDS. Transgenic plants have emerged as a promising technology to create recombinant biopharmaceutical proteins and vaccines. They offer a spectrum of exclusive advantages, and so their potential to be used as bioreactors for the production of therapeutic molecules is a current area of research intensively explored. Plant systems produce full-length mammalian proteins that appear to be processed similarly to their native counterpart with appropriate folding, assembly, and post-translational modifications. In fact, a wide variety of complex and valuable foreign proteins can be expressed efficiently in transgenic plants [1]. Production of recombinant proteins in plants offers economic advantages. It has been estimated that the cost of producing proteins in transgenic plants may be 100 fold lower than in transgenic animals or mammalian cell cultures, and the possibility of using the plant tissue as a carrier for oral delivery could also diminish the expensive step of recombinant protein purification. The use of plants as an expression system for recombinant protein production would be at least as economical as traditional industrial facilities (by fermentation processes or bioreactor systems). Some important potential advantages of producing recombinant proteins in plants for vaccine development include: production as well as storage can occur near the site of use; the heat stable formulations eliminate the need for refrigeration; and the need for hypodermic delivery (needles) can be also eliminated. These criteria indicate that a plant-based production system is a promising technology for the generation of easily distributed, affordable HIV vaccines. Additionally, one of the most obvious benefits of using plants as protein expression systems is the potential for scale up. Potentially vast amounts of recombinant protein could be produced simply by increasing the planting area. One of the major obstacles to recombinant protein production in plants is the low level of protein expression. At least 45 antigens have been successfully expressed in plants but their levels of expression are low, between 0.0005 – 0.3 % of total soluble protein (TSP) [1]. Moreover, because a protein purification step is needed for the final product of a plant-based production system, the final protein yield is therefore diminished. In this regard it has been estimated that a protein expression level compatible with purification technologies could be represented by 1% TSP [2]. Therefore, improving the foreign protein production yield in plants is a crucial objective that will have a significant impact on the economic feasibility of plants as bioreactors. Different strategies - use of novel promoters, codon optimization, improvement of protein stability, targeting of recombinant proteins to intracellular compartments, and improvement of downstream processing technologies - have been used to improve production. However, increasing the yield of foreign protein in plants is still a major goal of plant biotechnology, for which further optimization strategies are required. Experimental design and engineering of HIV-1 antigen-antibody fusion molecule One of the most important HIV antigens likely to form part of any HIV vaccine is the HIV-1 p24 capsid protein. It belongs to the group of proteins known as Gag proteins and constitutes one of the major structural proteins of HIV. Studies have shown cross-clade antibody responses against conserved epitopes of Gag in HIV infected individuals, and the absence of anti-Gag antibodies is indicative of disease progression. Moreover, T-cell immune responses are probably the most important protective mechanism against HIV, and the HIV-1 p24 antigen is the reported target of T-cell immune responses in infected individuals [3,4]. Therefore, efforts are being made to develop more efficient and feasible expression strategies for production and therapeutic use of recombinant p24. The HIV-1 p24 protein has been recently expressed in tobacco plants by different strategies. However, although research demonstrates that HIV-1 p24 can be successfully expressed in tobacco plants, consistently low expression levels of the p24 antigen in stably transformed plants are reported [5]. Mammalian immunoglobulin (Ig) is the only class of molecules reported to reliably reach high expression levels in transgenic plants (IgG antibodies, 1% TSP, and the secretory immunoglobulins, IgA, 5%-8% TSP) [6,7]. There is a significant difference in protein expression levels between monomeric antigens and polymeric Ig in plants; the reason for this difference is still not determined. Nevertheless, we decided to examine the potential of antibody sequences to enhance recombinant antigen expression levels in transgenic plants. Furthermore, since our studies intended to explore vaccine candidates in transgenic plants, we decided to investigate the design and engineering of an antigen-antibody fusion molecule capable of retaining the immunogenicity of the antigen fusion partner while incorporating the functional components of the antibody fusion partner. We established two molecular approaches for the expression and production of the HIV-1 p24 antigen in transgenic tobacco plants. First, as a control, we engineered the unmodified single HIV-1 p24 gene (p24) to be expressed under the control of the constitutive CaMV 35S promoter and its translation product targeted to the plant endomembrane system. Second, we engineered the HIV-1 p24 antigen-antibody fusion molecule. Immunoglobulins are polymeric molecules constituted by four monomeric chains: two identical heavy chains and two identical light chains. Each chain, heavy and light, possesses two different regions: one variable region involved in the recognition of antigen; and one constant region required for assembly of the chains. Moreover, the heavy chain constant region is also involved in activation of immune effector functions. Thus, in this second strategy, the HIV-1 p24 antigen was fused to the Ca2 and Ca3 constant region domains of a human immunoglobulin (IgA) heavy chain (p24/Ca2-Ca3) and its expression in tobacco plants was investigated. After sequencing analysis, both p24 genetic constructs were separately cloned into a pMON530 plant expression vector. A specific mouse IgG 5' leader sequence had been previously included upstream of each transgene into the vector in order to direct the recombinant protein to the plant endomembrane system. Subsequently, each genetic construct was individually transferred into Agrobacterium tumefaciens strain LBA4404 by electroporation. Transformation of Nicotiana tabacum (var. Xantii) plants was made by co-cultivation with Agrobacterium transformants, and the effect of the random nature of Agrobacterium-mediated gene insertion on the protein expression levels was minimized by meticulously selecting the highest expresser transgenic plants of each construct. Enhancing HIV-1 p24 antigen expression by IgA heavy chain fusion partner Two genetic constructs, p24 and p24/Ca2-Ca3, were engineered for expression in tobacco plants. As a first step, after generation and selection on antibiotics of transgenic plants transformed with either one or the other chimeric construct, the expression of both plant-derived recombinant proteins, p24 or p24/Ca2-Ca3, was analyzed. By using transgenic plant protein extract and specific anti-HIV-1 p24 antibodies in ELISA and Western blot, the accumulation of correctly folded recombinant full-length HIV-1 p24 was demonstrated in both cases. The domains Ca2 and Ca3 are responsible for the dimerization of a-heavy chains in immunoglobulin A (IgA) molecules under natural conditions. In our study, the expression of the full-length p24/Ca2-Ca3 fusion molecule was also confirmed, and its assembly into dimer molecular form indicates that the IgA Ca2-Ca3 domains fragment retains its native capability to assemble when expressed in plants as the p24/Ca2-Ca3 fusion partner. The HIV-1 p24 gene DNA sequence was identical in both constructs, and the plant codon usage was not optimized in either case. Thus, since the p24 antigen was efficiently expressed in tobacco plants by both strategies, the next step was to investigate the level of HIV-1 p24 expression in each case. An important difference in the p24 protein expression levels was observed when the HIV-1 p24 gene was expressed after fusion with the human IgA Ca2-Ca3 heavy chain sequence. A significant increase of up to 13-fold in the overall expression levels of p24 antigen in p24/Ca2-Ca3 transgenic plants was achieved (1.4% TSP) compared to those of p24 antigen when the HIV-1 p24 gene was expressed alone (0.1% TSP) in transgenic tobacco plants. Plants are very efficient at producing immunoglobulins, probably because the endomembrane system of plant and mammalian cells are organized in an identical manner. In addition, plant chaperones homologous to mammalian chaperones have been described within the plant endoplasmic reticulum (ER), and their interaction with Ig chains determines the efficiency of protein folding and assembly [8]. However, although both Ig heavy and light chains can be expressed individually in plants, enhancement of recombinant Ig expression levels has been reported when light and heavy chains are co-expressed in transgenic plants [6]. These results suggest that the assembly status of the molecule is a determinant of stability, and accordingly, the observation of p24/Ca2-Ca3 homodimers during our study suggests that the addition of Ca2 and Ca3 domains in the p24/Ca2-Ca3 fusion molecule may confer some structural advantages in terms of recombinant protein stability expressed in plants. At the same time, sub-cellular targeting plays an important role in determining the yield of recombinant protein, as it strongly influences the processes of protein folding, assembly, and post-translational modifications. Antibodies targeted to the secretory system usually accumulate to significantly higher levels than those of antibodies expressed in the cytosol. Moreover, the stability of antibodies is lower in the apoplast than in the lumen of ER. In our study, sub-cellular trafficking of both p24 and p24/Ca2-Ca3 proteins was also analyzed by immunoprecipitation and pulse-chain experiments in transgenic tobacco protoplasts. Our results demonstrated that HIV-1 p24 recombinant protein is efficiently secreted to the extra-cellular space when it is expressed alone. Conversely, HIV-1 p24 fused to human heavy chain Ca2-Ca3 domains is retained inside the cell. Proteins that accumulate in the secretory system are secreted into the apoplast in the absence of further targeting information. IgA antibodies accumulate predominantly within the plant endomembrane system and, in part, are targeted to vacuoles. The presence of a cryptic sorting signal in the IgA Ca3 tailpiece has been identified as an element responsible for this vacuolar targeting [9]. However, despite this sub-cellular targeting, IgA is expressed at high levels in plants and, indeed, at higher levels than other (IgG) antibodies, which are secreted to the extracellular space. In accord with these observations, we confirmed that p24/Ca2-Ca3 fusion molecule contains that same sorting signal, and taken together, our results indicate that the addition of IgA Ca2-Ca3 sequence may divert the recombinant HIV-1 p24 antigen to a different sub-cellular compartment than the HIV-1 p24 expressed alone. For immunogenicity testing of the p24 antigen in the context of an antigen-antibody fusion molecule, eight groups of five BALB/c (H-2d) mice were subcutaneously immunized at day 0 with different doses (3 µg , 10 µg, and 30 µg) of either purified plant-based p24/Ca2-Ca3 fusion protein or E. coli p24-His (as a positive control). A group injected with PBS buffer was used as a negative control. In all cases, alum was included as an adjuvant. Mice were boosted at 3 and 8 weeks, and samples collected at 0, 3, 8, and 11 weeks. Importantly, serum analyses revealed that plant-derived p24 is immunogenic in mice when expressed as the p24/Ca2-Ca3 fusion molecule, under a dose-dependant response, with highest titers after priming with 10 µg of recombinant protein. Furthermore, T-cell epitopes were conserved in plant-derived HIV-1 p24, as T-cell responses were elicited in mice against both plant-derived as well as bacteria-derived recombinant p24 antigen. In terms of vaccine development, we foresee some applications where it may be preferable to retain the Ig sequence on the final recombinant fusion protein. IgA is the most abundantly Ig produced in the body. It is localized on both sera and mucosal surfaces. The main route for the HIV infection to be contracted in more than 90% of HIV-infected individuals is via the mucosal surfaces of the genital tract or rectum, or through breastfeeding, and studies have shown that an important implication of secretory IgA, which constitutes the main class of antibody in this area, is a protective mucosal immune response against HIV. More recently, the so-called IgA Fc a-receptors have been defined as the most likely candidate to initiate potent effector immune functions upon binding to serum IgA through heavy chain constant domains [10]. In this context, dimers of the p24/Ca2-Ca3 fusion molecule may bind to Fc a-receptors to trigger a specific immune response. We have demonstrated that Ig fusion partners can be used as an alternative strategy for enhancing recombinant antigen expression in plants. There are still other factors to be considered before this technology can be ready for practical use. However, the antigen-antibody fusion strategy might lead to a new technology with important implications for both the economic viability of using plants as bioreactors for recombinant protein production and the development of a strategy to design new vaccines with enhanced specific immunological properties against HIV and other diseases. References Arntzen C et al. (2005). Plant-derived vaccines and antibodies: potential and limitations. Vaccine 23(15), 1753-6. Kusnadi AR et al. (1998). Processing of transgenic corn seed and its effect on the recovery of recombinant betaglucuronidase. Biotechnol. 60, 44-52. Dyer WB et al. (2002). Correlates of antiviral immune restoration in acute and chronic HIV type 1 infection: sustained viral suppression and normalization of T cell subsets. Aids Res human Retroviruses 18, 999-1010. McMichel AJ et al. (2001). Cellular immune responses to HIV. Nature 410, 980-7. Zhang GG et al. (2002). Production of HIV-1 p24 protein in transgenic tobacco plants. Mol. Biotechnol. 20, 131-136. Hiatt A et al. (1989) Production of antibodies in transgenic plants. Nature 342, 76-78 Ma JK et al. (1995). Generation and assembly of secretory antibodies in plants. Science 268, 716-719. Nuttall J et al. (2002). ER-resident chaperone interactions with recombinant antibodies in transgenic plants. Eur J Biochem 269, 6042-51. Hadlington et al. (2003). The C-terminal extension of a hybrid immunoglobulin A/G heavy chain is responsible for its Golgi-mediated sorting to the vacuole. Mol. Biol. Cell 14, 2592-2602. Otten MA et al. (2004). The Fc receptor for IgA (Fc-RI, CD89), Immunol. Lett. 92, 23-31. Enditem