www.checkbiotech.org ; www.czu.cz ; www.raupp.info

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, January 2007 by
Patricia Obregon.

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 ?g , 10 ?g,
and 30 ?g) 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 ?g 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.

[www.isb.vt.edu]

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