Introduction

The acquired immune deficiency syndrome (AIDS) caused by the human immunodeficiency virus (HIV) is one of the most significant diseases worldwide. In 2008 the UNAIDS had estimated that 33.4 million people were HIV-positive and global spread of HIV-AIDS has reached pandemic proportions. The HIV belongs to the genus Lentivirus and the family Retroviridae. These enveloped RNA viruses produce characteristically slow, progressive infections. Lentivirus replication depends on the presence of an active reverse transcriptase responsible for the transformation of the viral RNA genome into a proviral DNA copy that integrates itself into the host cell chromosome. The provirus is eventually transcribed into a set of mRNAs that encode the viral proteins and progeny genomic RNA. Two types of HIV have been described: HIV-1 and HIV-2. Based in part on genetic variation of the surface envelope glycoproteins, HIV-1 isolates fall into three groups—M (Major/Main), N (Non-M, Non-O/New) and O (Outlier)—of which group M is the most common. Group M is subdivided into several subtypes or clades (A–D, F–H, J and K), of which B is the most common in the Western world and subtype C is primarily found in India, China and sub-Saharan Africa. The remaining subtypes, as well as HIV-1 variants with characteristics of several different subtypes (so-called circulating recombinant forms), are mainly spread throughout Africa (Karlsson Hedestam et al. 2008).

The introduction of the highly active antiretroviral treatment (HAART) in industrialized countries had resulted in a considerably reduced disease progression to AIDS and has transformed HIV infection from a lethal disease to an effectively manageable chronic disease (Palella et al. 1998; Walensky et al. 2006). Although many of the newer HAART regimens show very low rates of toxicity, cumulative effects over decades of treatment are not discarded (Mallon 2007; Richman et al. 2009). Moreover, emergence of multiclass drug-resistance in HIV in antiretroviral-treated individuals represents an imminent risk for current treatment especially in developing countries (Gottlieb et al. 2009). For these reasons, vaccination is still considered the ideal solution against AIDS epidemic.

Many attempts to develop an effective vaccine have been carried out, including attenuated and inactivated virus, protein subunits, synthetic peptides, DNA vaccines, viral vectors expressing HIV-1 genes, among others (Gamble and Matthews 2010). However, the first two approaches were essentially discontinued because they did not retain their immunogenic properties, resulting in a low protective efficacy. Moreover, biosafety issues have also been involved in circumventing their advancement (Girard et al. 2006). For this reason, the current rational HIV immunogen design is focused on developing subunit vaccines based on immunogenic HIV chimeric proteins, capable of eliciting broad humoral and cytotoxic CD8+ responses (Walker et al. 2011; Graham et al. 2010), which should ideally protect against HIV. Regarding induction of humoral immunity, the Env complex is the only identified target able to elicit neutralizing antibodies (NAbs) which can block the viral entry. These NAbs have been evaluated in passive immunization schemes on macaques (Klasse et al. 2011; Pantophlet and Burton 2006). Other structural and early components (e.g., Gag, Tat or Nef) have showed to be immunogenic, but the resulting Abs are not neutralizing because the target proteins are inaccessible in the virion (Mascola et al. 1999). These targets are however of relevance in the induction of cellular responses, which eventually can suppress viral load.

In this context, three vaccine concepts have completed clinical efficacy studies so far, two of them with negative results (Barouch and Korber 2010). In the case of the Env gp120 vaccine–AIDSVAX, a total of 5,403 volunteers (5095 men and 308 women) were evaluated. This vaccine did not prevent HIV-1 acquisition: infection rates were 6.7% in 3,598 vaccinees and 7.0% in 1,805 placebo recipients (Flynn et al. 2005). On the other hand, the AIDSVAX B/E vaccine contains 2 rgp120 HIV-1 envelope antigens: one from a CXCR4-dependent laboratory-adapted subtype B strain (MN), and other from a CCR5-dependent primary subtype CRF01_AE isolate (A244). A total of 2,546 injection drug users were enrolled. There were no differences between the vaccine and placebo arms and vaccine efficacy was estimated at 0.1% (Pitisuttithum et al. 2006). The third one consisted of a Phase III clinical trial in Thailand, where a recombinant canarypox vector expressing Envelope, Gag, and segments of Pol and Nef proteins from HIV-1 subtype B/E was evaluated in combination with a recombinant HIV-1 B/E gp120 formulated in alum adjuvant. Interestingly, this approach showed a reduction in the risk of HIV infection in a community-based population with largely heterosexual risk, showing borderline efficacy, with no effect on HIV-1 viral load (Rerks-Ngarm et al. 2009).

Due to these limited results on clinical trials, the design of HIV-1 immunogens capable of inducing with a few doses high-titer antibodies that neutralize a broad spectrum of HIV-1 primary isolates is a major priority for HIV-1 vaccine development. Taking into consideration that the vast majority of infected people live in sub-Saharan Africa or developing countries, an affordable vaccine formulation is also required. To date, plant-based vaccines have allowed the assessment of many immunization models (Yusibov et al. 2011). The trend of using plant-based vaccines that are able to prime the immune system resulting on mucosal responses directed against infectious agents, has been evaluated in several studies focusing on pathogens that primarily infect mucosal surfaces (Rosales-Mendoza et al. 2009). This approach is appropriate for HIV since the mucosa is the main route of the virus entry and therefore antibodies directed against specific HIV antigens could block the virus transcytosis in the epithelium and thus prevent infection of the CD4+ T cells. Interestingly, systemic immune responses can also be induced by this route. This topic of plant-based vaccines on HIV fight has been explored over the last two decades and the present review summarizes these efforts in the following sections and also presents some of the gp120-based approaches performed by our group. The HIV antigens that constitute promising candidates on the development of vaccine are divided in three groups: early antigens, structural proteins and envelop proteins.

An overview of the attempts of developing plant-based HIV vaccine models

Early antigens: tat and nef as special cases

Early antigen refers to those proteins that are expressed in the early stage of the virus life cycle. Six early proteins are present in the HIV: Tat, Rev, Vpu, Vif, Nef, and Vpr. Soluble HIV-1 proteins such as Nef, Tat, and Vpr and Vpu have been detected in serum of HIV-1 infected patients, possibly released by infected/apoptotic cells. Thus they are thought to be able to enter macrophages and to modulate both cellular machinery and viral transcription (Herbein et al. 2010; see Fig. 1a).

Fig. 1
figure 1

a Schematic representation of the HIV components extensively studied in the vaccine development field, grouped in early proteins, including the accessory proteins (blue squares), structural proteins (green) and envelop proteins (red). Function of each component are summarized (Li et al. 2005; Klein et al. 2007). b HIV components used so far as targets of plant-based vaccine models resulting in immunogenic activity. p.o. oral, i.n. intranasal, i.m. intramuscular, i.p. intraperitoneal, CP coat protein, CPMV Cow pea mosaic virus, CTB cholera toxin B subunit, AMV Alfalfa mosaic virus

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Nef is a 27 kDa protein which is expressed in the early stage of the virus life cycle. It down-regulates the cell surface expression of CD4, CD28, and MHC class I (Lundquist et al. 2002; Yang et al. 2002) conferring to Nef a key role in pathogenesis. This HIV component has showed to induce cellular responses in a number of experimental models, which in conjunction with the role of Tat on the viral cycle, justifies its use as an HIV immunogen (Rolland et al. 2011; Shen et al. 2011).

Tat is a small protein essential for the efficient transcription of viral genes and for viral replication. This protein potently transactivates LTR-driven transcription, resulting in a remarkable increase of viral gene expression (Kessler and Mathews 1992; Zhou and Sharp 1995). This HIV component is able of eliciting cellular responses, which along with its role on HIV replication convert this antigen in a promising HIV immunogen (Ensoli et al. 2010).

These early components have been expressed in plants as full-length proteins and also as fusion proteins to improve their accumulation or enhancing immunogenicity (Table 1).

Table 1 Description of the reported HIV candidate plant-based vaccines targeting early HIV proteins
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On the other hand, Ramírez et al. (2007) have expressed the full length Tat in tomato proving the immunogenicity of the tomato-derived protein via oral, intraperitoneal, or intramuscular administration routes. In addition, the relevance of the immune responses was reflected in a Tat neutralization assay, where a reduced transactivation in HL3T1 HeLa cell was observed in the case of the treated animals. Further studies performed in nonhuman primates to determine the reproducibility of these observations in a more relevant experimental model would be of great interest in this field.

So these Tat-based vaccines seem to be relevant as they have proven not only to be safe and immunogenic in preclinical models, but also effective in controlling virus replication and blocking the onset of the disease in monkeys (Goldstein et al. 2000; Maggiorella et al. 2004). However, this approach is therapeutic rather than preventive (Ensoli et al. 2010): this limitation has been evidenced in trials performed in monkeys where only a limited protection against the disease has been reported (Cafaro et al. 1999). Additionally, another important factor to be considered is related to the difficulty of inducing effective cellular immune responses under the conventional plant-based vaccines approach, since the natural antigen presentation does not occur.

Structural proteins: p24

The structural viral proteins comprise the components of the mature assembled virus particles such as the nucleocapsid core proteins (Gag proteins). The protein Pr55Gag is coded by the gag gene and is cleaved by the viral protease to the mature Gag proteins: matrix (p17), capsid (p24), nucleocapsid (p7) and p6 (see Fig. 1a). The p24 protein is an important early marker of HIV infection and has been shown to induce cellular and humoral responses. This core protein is the target of T-cell immune responses in both primary and chronically infected individuals. It has been reported that the absence of anti-Gag antibodies is indicative of disease progression (Montroni et al. 1992; Reddy et al. 1992; Benson et al. 1999; Dyer et al. 2002; Novitsky et al. 2003). On the other hand, p17 facilitates the intra membrane associations necessary for viral assembly and release as well as being involved in the transport of the viral pre-integration complex into the nucleus. It is well documented that the risk of AIDS is greatly increased in individuals with falling titres of p24 antibodies, suggesting that high anti-p24 antibody titters might be necessary to maintain a disease free status (Cheingsong-Popov et al. 1991; Burkinsky et al. 1993; Novitsky et al. 2003). These proteins are therefore potential candidates for the development of HIV subunit vaccines. Table 2 summarizes the reports related to the expression of HIV structural proteins in plants.

Table 2 Description of the reported HIV candidate plant-based vaccines targeting structural HIV proteins
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A remarkable report consists of the expression of p24 in Nicotiana benthamiana plants (Meyers et al. 2008), where the heterologous protein was expressed by both transient and stable approaches and the authors observed an immunogenic activity in a prime-boost scheme where animal were primed with a DNA vaccine and the plant-derived p24 was used as boost by the intramuscular (i.m.) route. Despite this immunological data, considering that the expected final application of plant-based vaccines consists on the development of oral formulations, the immune response induced by oral administration of this plant-derived antigen remains to be studied.

Env proteins gp41 and gp120

The envelope glycoproteins resides on the surface of the virus and plays a crucial role in HIV infection. The Env glycoprotein (gp160) engages the HIV-1 receptor and co-receptors, mediating the virus entry into host cells, which is a very important step in the viral life cycle. The gp160 is cleavage in two subunits: gp120 and gp41, which constitute the primary targets for NAbs (Doms 2004).

Since the gp120 participates in the interaction between the receptors of the host cell and the virus surface, a number of strategies are being pursued to design immunogens capable of eliciting broadly NAbs to this antigen (Pantophlet and Burton 2006). Elements of the third variable domain from gp120 (V3) are important conformational determinants that acts as binding sites to the co-receptors CCR5 and CXCR4 at the host cells (Haynes et al. 2006) and thus these constitute crucial targets for blocking the virus entry.

One of the challenging issues involves the high rate of mutation of the env gene which leads to changes in its antigenic properties and eventually to the escape from the elicited NAbs. This fact represents a major challenge for the development of HIV vaccines. Interestingly, it has been shown that broadly neutralizing antibody responses against Env are developed in a large percentage of HIV-infected individuals (Simek et al. 2009; Stamatatos et al. 2009) and these have been characterized as candidate for the development of passive immunity-based therapies. Importantly, animal studies have demonstrated complete protection by passive immunization with such antibodies (Burke and Barnett 2007). The enveloped protein gp41 mediates the fusion of membranes and permits the release of viral genome into the host cells, making it another relevant target for vaccine design. Many gp41 epitopes have been well characterized and some are able to elicit humoral responses that inhibit viral trancytosis and membrane fusion, which make them promising candidates for vaccine formulation (Tudor et al. 2009). Conveniently, these gp41 epitopes, especially the membrane proximal region (MPR) area, are more conserved that the V3 of gp120, thus having a higher ability of inducing broad protective responses (Dimitrov et al. 2007; Frey et al. 2008; Luftig et al. 2006).

To date some reports on the expression of Env epitopes in plants have been published (Table 3). Most of them comprise the use of chimeric proteins formed by carrier proteins, such as virus-like particles (VLPs), and chimeric virus or the cholera toxin B subunit (CTB). However, studies on envelope antigens are limited to a few reports. For example, pentameric protein CTB was fused to the V3 envelope antigens and expressed in stably transformed potato plants. Although the CTB-gp120 was assembled into pentamers and retained the antigenic properties of both components, its immunogenicity remains to be tested (Kim et al. 2004). In another report Matoba et al. (2004) describe the production of a fusion protein comprising CTB and the P1 peptide from gp41 (aa 649–684) in transiently transformed Nicotiana benthamiana plants and E. coli. Both plant and bacterial derived CTB-P1 were able to trigger serum IgG and mucosal IgA responses when administered by the in route in mice, suggesting that plant-derived CTB-P1 have the potential as mucosal immunogen against HIV (Matoba et al. 2009). In addition, the bacterial CTB-P1 has showed to induce transcytosis-neutralizing antibodies, which opens interesting perspectives related to this antigen. Thus, this particular attempt is likely to have future implications in the field of effective mucosal vaccines since CTB is recognized as a highly effective vaccine adjuvant/carrier capable of delivering unrelated antigens, inducing both mucosal and systemic immunity (Johansson et al. 1998). Eventhough these results are promising, the HIV neutralizing potential in mice and the oral immunogenicity of the raw plant material remain to be tested.

Table 3 Description of the reported HIV candidate plant-based vaccines targeting envelop HIV proteins
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Using a chimeric virus approach, a gp41 domain (aa 731–752) has been expressed as fusion along with the coat protein from the Cowpea mosaic virus (CPMV). The resulting chimeric virus was able to elicit strong serum neutralizing antibody response when subcutaneously administered (McLain et al. 1996). The oral immunogenicity of the raw plant material remains to be assessed, however. The use of chimeric virus is an attractive focus since they possess a high immunogenicity and can present heterologous HIV neutralizing epitopes, avoiding the use of the native viral envelope glycoproteins, which tend to be poorly immunogenic and unstable, and can induce responses against a set of non-neutralizing epitopes (Pejchal and Wilson 2010).

Under this view, the following section describes the efforts that are being conducted by our research group.

Implications of C4 and V3-based polypeptides on HIV vaccine design

Because of the extreme variability of HIV-1, the most effective vaccines against this virus are expected to be those that target invariant virus-surface antigens. Moreover, it would be preferable to identify and include in the vaccine formulation antigenic linear epitopes able to induce broadly neutralizing antibodies. Antibody responses against specific regions of gp120 are required to block the initial binding of the virus to target cells. However, gp120 appears to be quite elusive insofar as it has the apparent ability to change conformation on binding to CD4, its cellular receptor, and, as the conformational changes occur, antibody epitopes also would appear or disappear. From our current knowledge, therein lies the current challenge: to make immunogenic, conformationally constrained fragments of the highly conserved elements in gp120 that exist prior to CD4 binding to be able to elicit antibodies capable to block the binding of gp120 to CD4. In addition, it is desirable to do this without exposing the chemokine receptor ligands in gp120, which are exposed when gp120 binds to CD4. The exposure of the chemokine receptor ligands in gp120, such as V3, can mediate the infection of cells that do not express CD4 but do express the chemokine receptor (Robey and Robert-Guroff 2001). It is therefore also important to develop strategies to induce anti-V3 antibodies. The fourth conserved domain of gp120 is believed to be a major part necessary for binding to CD4. A α-helix structure is suggested for this domain (Robey et al. 1995).

The epitopes that are known to induce broadly neutralizing Abs include the membrane proximal external region of gp41, and the following regions from gp120: the CD4 binding site, complex glycans, the CD4-induced epitope in and around of the bridging sheet, and the V3 loop (Zolla-Pazner 2004).

Synthetic peptides comprising the V3 loop and C4 domain from gp120, designated C4V3 possess immunogenic properties, which is probably explained by the role of C4 in the CD4 binding event (Graham et al. 2010). Synthetic peptides constitute the straightforward approach to characterize these candidates; however, this approach limits the potential for clinical trials and global vaccination. Varona-Santos et al. (2006) have reported the design of a C4V3 polypeptide based on the MN HIV isolate, constituted by the 17 aa sequence EKQIINMWQEVGKAMYA of the C4 domain and the 23 aa sequence TRPNYNKRKRIHIGPGRAFYTTK of the V3 loop. This recombinant protein (rC4V3) produced in E. coli at convenient yields (75 mg/culture liter) showed to be immunogenic in Balb/c when administered by the intranasal (i.n.) and i.m. routes without the need of adjuvants. Based on these results, the rC4V3 protein is postulated as a promising tool on HIV immunization studies.

Our group is currently exploring the feasibility of producing C4V3-based polypeptides in plant systems as an immunization alternative in the fight to HIV. Transplastomic tobacco plants have been developed to produce a functional C4V3 polypeptide that showed convenient antigenic and immunogenic properties in mice (see Fig. 2a). Expression levels allowed to immunize with low quantities of freeze-dried tobacco (50 mg containing 15 μg of C4V3), minimizing its toxic effects on mice. Immunogenicity was determined dosing mice orally with ≈15 μg of plant-derived C4V3. The scheme included four oral doses of freeze-dried tobacco leaves previously grounded and re-suspended in PBS right before oral administration. No adjuvants were used under this approach. Significant humoral responses were elicited at the mucosal and systemic level. This assay showed that C4V3 produced in tobacco is immunogenic and therefore would serve for the assessment of the elicitation of neutralizing antibodies in further studies. Moreover, based on flow cytometry proliferation and intracellular IFN-γ production assays, it is likely that the tobacco C4V3 protein is capable of triggering CD4+ cellular responses. These evidences therefore support the viability of using plant chloroplasts as biofactories for HIV candidate vaccines and could serve as an important tool for the development of a plant-based candidate vaccine against HIV (unpublished). However, since tobacco leaves contain large amounts of phenolics and toxic alkaloids, such as nicotine, the next step was the expression of the C4V3 polypeptide in edible crops so that detail immunization experiments with edible materials could be accomplished. On the other hand, another important aspect was to study if the inclusion of tandem repeats of V3 sequences from different HIV isolates can result in a wider immune coverage. Under this panorama, the next approach pursued by our group included the expansion of a protein based C4V3 configuration but adding five different V3 sequences to broaden the coverage of the humoral response to be elicited. This polypeptide, designated C4(V3)6, has been expressed in lettuce plants by means of nuclear transformation techniques and had shown to be compatible with this plant expression system. It is also immunogenic by the oral route in mice, following the same scheme used for the tobacco-derived C4V3 (see Fig. 2b, unpublished). These lines of such edible crop represent a step forward in the development of HIV oral vaccine formulations (Table 4).

Fig. 2
figure 2

a C4V3-producing tobacco line (TT) showing the SpeR phenotype in contrast with the chlorotic plantlets developed from the wild type seeds (WT). b C4(V3)6-producing lettuce line (LT) showing its KanR phenotype characterized by a higher rate of growth instead the slow growing ones developed from the wild type seeds (WT). These lines have showed immunogenic activity in mice when administered by the oral route and are candidates to be evaluated in non human primates to assess the elicitation of neutralizing antibodies

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Table 4 Summary of the plant lines developed by our group to target the expression of gp120 epitopes
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The road ahead is still long and the consideration of assessing the neutralizing ability of the elicited humoral response is a priority. On the other hand, the assessment of proteins carrying a wide set of epitopes from additional and relevant HIV proteins should also be explored. Immunogenic properties of these new chimeric proteins are nonetheless basically unpredictable and should be assessed individually for each candidate.

Chimeric proteins comprising different types of HIV proteins

Some groups have attempted to produce chimeric antigens comprising different types of HIV proteins (Table 5). This is considered the ideal approach in the development of highly efficient vaccines since several advantages are implied: (1) formulations with a wide coverage can be produced in a single protein by including a set of epitopes from different isolates, (2) it allows for the exclusion of non immunogenic regions which may mask the presentation of the target neutralizing epitopes, (3) sequences with adjuvant activity can be included, (4) formulation is easier when a the vaccine is based in a single mutiepitopic protein.

Table 5 Description of the reported HIV candidate plant-based vaccines targeting different types of HIV proteins
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One of the most advanced approach was reported by Zhou et al. (2008), based on the expression of the capsid protein p24 fused to Nef (p24-Nef), which were accumulated at high levels (40% TSP). Gonzalez-Rabade et al. (2011) recently investigated the immunogenicity in mice of both p24 and p24-Nef proteins and found that they were immunogenic when administered by the subcutaneous (s.c.) route. Oral immunization with chloroplast-derived p24-Nef was capable to enhance the immune response when used as boost after a s.c. priming. It is considered of critical importance to assess the oral immunogenicity of the plant-derived antigen per se in order to determine the feasibility of these plants to serve as an effective oral immunogen.

Another one of the most advanced approaches has consisted in the expression in tomato plants of a TBI multiepitopic sequence, comprising sequences from gp41 and p24, fused to the surface antigen of the Hepatitits B Virus (TBI-HBsAg). Interestingly this configuration achieved the induction of humoral responses when administered by the oral (p.o.) and intraperitoneal (i.p.) routes (Shchelkunov et al. 2006). The neutralizing potential of the elicited response remains to be tested in order to generate perspectives related to performing clinical trials.

Perspectives

Expanding immune characterization of plant-based vaccines

The recent advances in nonhuman primate models, neutralizing antibody design and the urgent need to conduct more clinical efficacy trials could impulse strength to the plant-based vaccine development. Induction of neutralizing antibodies is widely considered a critical parameter of protection for many licensed vaccines, but no HIV immunogen induces sterilizing immunity. Given our current state of knowledge, testing more vaccine formulations with distinct immunogenic profiles will be greatly informative.

It is clear that after proving the concept of using plants as vaccine biofactories, a detailed immunological characterization remains to be performed for the majority of the reported approaches considering this a priority on the field. Proving immunogenicity on mice but also in a more relevant model such as monkeys as well as performing neutralization assays, are critical procedures to estimate the efficacy of the proposed vaccines.

Triggering mucosal immune responses

The most common routes of HIV transmission are the genitourinary and rectal mucosa where HIV enters across epithelial cells (Hladik and Hope 2009). Since mucosal immune responses can be efficiently induced by the administration of vaccines onto mucosal surfaces, these offer the possibility of leading to HIV immunity (Levine 2000; Lamm 1997).

Unlike parenteral immunization, vaccination by the oral route, i.e. passage of the vaccine through the stomach and the intestine, has a promising potential as a delivery site for prospective HIV vaccines that may induce both systemic and mucosal immune responses (Hone et al. 1996; Stevens et al. 2004). This is part of the rationale of using edible plants as putative HIV oral vaccines.

The use of appropriate adjuvants to favour the priming of the mucosal immune system should be considered. Cholera toxin and the heat labile toxin from E. coli are considered typical mucosal adjuvants. However, this field of research should be directed towards assaying new molecules with recently proved adjuvanticity, such as some plant derived metabolites and the Bacillus thuringiensis Cry1Ac protoxin, among others (Vázquez et al. 1999; Guerrero and Moreno-Fierros 2007; Licciardi and Underwood 2011). Molecular approaches to achieve the co-expression of the desired adjuvant in plants are currently available and can be used for this goal. For example, plastid transformation offers the ability to express different ORFs at the same time in the form of bicistrons, while some viral elements such as IRES from Tobacco Mosaic Virus have showed to allow the expression of two nuclear encoded proteins trough a bicistron (Ali et al. 2010). The design of fusion proteins comprising a protein with adjuvant properties and the antigenic target seems to represent another viable approach. However, retention of the functionality of both components is basically unpredictable and should be studied case by case.

Another important issue that has been studied over the last two decades is the achievement of sufficiently high levels of accumulation of the target protein, which is crucial for the formulation of oral vaccines. In this regard, chloroplasts transformation has proved to serve as a convenient platform with remarkable advantages. The variable expression levels observed under the nuclear transformation approaches are considered a consequence of position effects and gene silencing. Targeting the integration of the transgene at specific sites into the chloroplast genomes eliminates the position effects. In addition, gene silencing has not been observed in chloroplasts, even at high levels of transgene expression. Therefore, transplastomic approaches seem to be the most advantageous focus to afford a high and uniform immunogen accumulation (Daniell 2006).

Triggering cellular immune responses

Humoral and cellular responses are necessary for an effective anti-HIV vaccine because these allow the blocking of the virus entry, killing the initial infected cells or preventing viral replication. Eventhough antigens encapsulated in plant tissues play an important role in oral vaccination leading to local humoral responses, one of the main disadvantages of these vaccines consists in the poor cellular response generated due to the fact that the antigen is not present in their natural context. Therefore, approaches to trigger such type of response could lead to an improvement of the formulations. This is however a challenge since this goal often required adjuvant and suitable delivery strategies.

In this context, dendritic cells are critical for the development and activation of T cells, and can be favored by adjuvants that are co-administered with the antigen. The role of innate immunity is essential for eliciting strong T cell responses, for example TLR activation can direct the antigen to different antigen presenting cells (APCs) and substantially increase the T cell immunogenicity of purified antigens. These approaches as well as the use of innovative immunization schemes such as prime boost schemes comprising different routes or antigens can generate superiorly T cell responses.

On the other hand, cytokines play an important role on polarization of the immune response thus they can act as vaccine adjuvants. In particular, the Interleukin-12 (IL-12) is a multifunctional cytokine that was originally described as a maturation factor for cytotoxic (T) lymphocytes and a cell stimulatory factor for natural killer cells. IL-12 is a heterodimeric glycoprotein composed of two disulfide-linked chains (p35 and p40) that has numerous effects on T and natural killer cells, resulting in enhancement of cytotoxic activity and induction of gamma interferon (IFN-γ) production. Thus IL-12 represents an important nexus for the development of type I cell-mediated immune responses (Villinger and Ansari 2010). These immunostimulatory properties of IL-12 have led to experimentation with its use as a vaccine adjuvant showing promising results in a number of models (Afonso et al. 1994; Arulanandam et al. 1999). In particular for HIV, the literature provides evidence about the role of IL-12 on inducing adjuvanted responses (Gupta et al. 2008; Villinger and Ansari 2010). Interestingly, mouse IL-12 has been produced in some plants including tomato where leaves and fruits accumulated up to 7.3 and 3.4 μg/g of fresh weight, respectively. A single chain human IL-12 was also expressed in tobacco reaching 40 ng/g of fresh weight in the leaves (Gutiérrez-Ortega et al. 2004). Murine IL-12 has also been expressed in tobacco hairy roots, which accumulated up to 5.3 mg/g FW when grown in a mist reactor (Liu et al. 2009). Importantly the functionality of plant-derived IL-12 has been also reported in both in vitro and in vivo assays (Gutiérrez-Ortega et al. 2004, 2005; Sánchez-Hernández et al. 2010).

These reports provide solid evidence on the hypothesis that co-expressing functional cytokines along with the antigen of interest would modulate the elicited immune response, favoring the elicitation of cellular responses (Soria-Guerra et al. 2011). Other cytokines of interest are the GM-CSF and the Flt-3L, which have showed to act as adjuvant on HIV DNA vaccination models (Xu et al. 2008).

Concluding remarks

Plant-based vaccines against HIV constitute a topic of great interest to researchers, which is reflected by the number of reports on expressing HIV antigen in plants. Advances on the production and characterization of putative protective proteins at the antigenic level have demonstrated the feasibility of this approach. In light of the current progress in this field it is also clear that a detailed immunological characterization for most of the explored antigens remains to be performed. However, since eliciting specific and broad cellular and humoral responses are required to prevent or reduce the severity of the HIV infection, it is therefore a mandatory necessity to evaluate new protein configurations in order to identify highly effective immunogens. On the other hand some goals such as coexpression of adjuvants will be facilitated by the transplastomic technologies leading to a substantial impact in this field. These advances will represent a step forward to the next preclinical steps where HIV neutralization may be evaluated. In conclusion, plant-based vaccines have raised a new perspective which constitutes an alternative that, in conjunction with the traditional approaches, might facilitate the fight against HIV.