Oral Vaccine Delivery: Can it Protect Against Non-mucosal Pathogens?

Lina Wang; Ross L Coppel


Expert Rev Vaccines. 2008;7(6):729-738. 

In This Article

Oral Vaccines Against Malaria

Malaria, an infectious disease caused by species of the parasite genus Plasmodium, is a major public-health problem, and the development of vaccines holds great promise for enhancing currently available control measures. Plasmodium has a complex life-cycle that includes multiple stages in both the mosquito vector and the human host. A number of Plasmodium antigens have been characterized and demonstrated to be potential vaccine candidates, including in pre-erythrocytic vaccines, asexual blood-stage vaccines and sexual-stage vaccines, and they are now in various stages of preclinical and clinical development for delivery by parenteral means.[31,32] Owing to the relative poverty and lack of infrastructure of many malaria endemic areas, particularly in sub-Saharan Africa, a successful immunization strategy will depend on cheap and scaleable methods of vaccine production, distribution and delivery. Affordable and effective vaccine formulations that can be delivered orally would significantly enhance widespread vaccine deployment.[4] In addition, the areas where malaria is endemic also have high prevalence of other infectious diseases, such as HIV and hepatitis B. Oral delivery can eliminate the potential transmission of infections caused by inadequately sterilized needles or needle reuse.

Oral delivery of malaria vaccines using live vectors has mainly used Salmonella, although the use of Lactococcus has also been reported recently ( Box 1 ).[33,34,35] Most of the studies have used circumsporozoite protein (CSP), which is the major surface protein on sporozoites and a component of the RTS,S parenteral vaccine now undergoing testing in widespread clinical trials.[36] An early study showed that oral immunization with recombinant Salmonella typhimurium expressing Plasmodium berghei CSP induced antigen-specific cell-mediated immunity and protected mice against sporozoite challenge in the absence of antisporozoite antibodies.[37] This immunity was mediated through the induction of specific CD8+ T cells, which were directed against the same peptide that was identified as the target of cytotoxic T lymphocytes (CTLs) induced by sporozoite immunization.[38] An in dependent study also demonstrated that S. typhimurium containing Plasmodium yoelii CSP induced CSP-specific CTLs in mice by oral administration.[39] In a small human trial, Salmonella typhi vaccine strain CVD 908 expressing Plasmodium falciparum CSP induced specific serum antibodies and CTLs.[40] However, these responses were modest and limited to only a fraction of the vaccinated subjects. Approaches have been taken to improve the immunogenicity of CSP delivered by Salmonella, including the use of genes optimized for prokaryotic codon usage and expression from genetically stabilized plasmids. Both have been shown to improve the levels of CSP expression.[41] Antigen-export systems provide another mechanism, either through directing secretion of the vaccine antigen.[42] or displaying the protein on the surface of Salmonella.[43]S. typhimurium secreting P. falciparum sporozoite surface protein 2, using secretion by the type I hemolysin system, was shown to induce higher immune responses following intranasal immunization compared with the nonsecreting construct.[42] Surface expression can be achieved by fusing the target antigen to the autotransporter MisL.[43] or the outer membrane protein A, as has been performed for three blood-stage antigens of P. falciparum: the serine-rich protein, the histidine-rich protein II and the ring-infected erythrocyte surface antigen.[44,45] These recombinant Salmonella were able to induce antigen-specific serum antibodies upon oral vaccination of mice.[43,44] Other strategies to enhance the expression and immunogenicity of Plasmodium antigens in Salmonella include fusion of the target antigen to tetanus toxin fragment C.[46,47] which is a highly stable foreign protein in Salmonella and can rescue expression of the fused foreign protein by rendering it resistant to bacterial pr-oteases.[48]

In general, Salmonella appears capable of delivering foreign antigens to induce a predominantly cell-mediated response, probably due to the fact that it delivers antigens to the intracellular space of host cells. This makes Salmonella more suitable for pre-erythrocytic stage antigens.[49] but less attractive for delivering asexual blood-stage and sexual-stage antigens, since immunity to these two stages is mediated predominantly or exclusively by humoral immune responses.[50,51] There has been one report in which recombinant Salmonella delivered an asexual blood-stage antigen: the N-terminal repetitive region of P. berghei merozoite surface protein (MSP)1.[52] A partial protective immune response was induced in the mouse model but it did not involve the production of detectable antibodies. Immunity to blood stages in the mouse model has shown a greater dependence on cellular immunity than has been demonstrated in humans, so the importance of this observation remains uncertain.

In contrast to the general perception that soluble proteins are poorly immunogenic by oral immunization, we have found that P. falciparum MSP4 (PfMSP4) and its homologue in P. yoelii (PyMSP4/5) are capable of inducing antigen-specific serum antibodies when delivered orally with CTB. The levels of antibodies were comparable to those achieved with parenteral immunization with the same antigen dose emulsified in Freund's adjuvant, and the immune responses induced orally with PyMSP4/5 could protect mice against a lethal challenge with P. yoelii parasites.[53] A second P. yoelii antigen, the C-terminal 19-kDa fragment of MSP1 (PyMSP119) was also immunogenic by oral immunization and offered a significant level of protection against parasite challenge.[54] Furthermore, a combination of PyMSP4/5 and PyMSP119 administered orally conferred markedly improved protection compared with either protein administered alone,[54] a phenomenon also seen following parenteral delivery of the same combination of antigens.[55] The source of antigen in these studies was from conventional recombinant production in E. coli. We have produced the PyMSP4/5 protein in transgenic tobacco and shown that the plant-made PyMSP4/5, when delivered orally, was able to boost the antibody responses induced by DNA vaccination.[56] However, the antibody levels were not high enough to protect the immunized mice against a lethal challenge with P. yoelii and further studies will be required to understand how the protein needs to be adapted to improve levels of immunogenicity and protective efficacy.

Reports from other groups have also demonstrated the effectiveness of noninvasive mucosal vaccination regimes by using different Plasmodium antigens. Arakawa et al. showed that recombinant Plasmodium vivax ookinete surface protein Pvs25 could induce transmission-blocking serum antibodies when given intranasally to mice with CT.[57] The level and isotype profile of the antibodies were comparable to those induced by intraperitoneal immunization with alum-absorbed Pvs25. Subsequently, the same authors confirmed that intranasal immunization of mice with the P. falciparum homologue of Pvs25, Pfs25, also induced strong serum antibodies when coadministered with CT, which could completely block parasite transmission to mosquitoes.[58] Intranasal immunization with MSP119, either from P. yoelii (PyMSP119) or P. vivax (PvMSP119), was able to induce serum antibodies and provided partial protection in the case of PyMSP119.[59,60] Although, in these studies, intranasal immunization was performed instead of oral delivery, they demonstrated the feasibility of mucosal vaccination regimes against non-mucosal infectious pathogens and justify the investigation of oral vaccines for the control of mucosa-unrelated diseases.

It is of interest that all the blood-stage and sexual-stage antigens described previously herein have a common structural feature: they all contain multiple disulfide bonds forming one or more EGF-like domains. For example, PfMSP4 (and its homologue PyMSP4/5) has one EGF-like domain, whereas MSP119 and Pfs25 have two and four EGF-like domains, respectively. It is possible that the tightly folded tertiary structure of these EGF-like domains makes them more resistant to intestinal proteolysis and results in the antigens being taken up and p-resented to the immune system in a relatively intact form. Alternatively, EGF-like domains may partake in protein-protein interactions, thereby facilitating their uptake into the mucosa-associated lymphoid tissues. This will need to be determined since encapsulation, an obvious strategy for antigen protection, may interfere with interaction with immune cells. Further work is also required to determine whether the capacity to induce effective immunity is confined to proteins of particular structures or whether it is possible for other Plasmodium proteins. Most workers in the field believe that any eventual subunit vaccine for malaria will contain multiple proteins from several life-cycle stages. The problems of formulating a multiprotein vaccine for parenteral use is very much greater than such a combination delivered orally. Problems of stability with adjuvant are less of a concern with oral formulation, although work is required to determine whether antigenic competition, a problem commonly seen with multiprotein parenteral immunization, also occurs with oral delivery.

Soluble proteins are, in general, poorly immunogenic by oral immunization. For example, soluble SPf66, a synthetic peptide consisting of four short sequences derived from two stages of the P. falciparum life-cycle (pre-erythrocytic and erythrocytic), failed to elicit detectable antibodies in mice by oral immunization.[61] When formulated with alum, it induced a partially protective immune response in Aotus monkeys; this protective effect was also reported in human volunteers, which was assessed through experimental challenge or natural exposure to parasites.[62] SPf66 was the first synthetic malaria vaccine to be tested in clinical trials. Its immunogenicity could be improved by encapsulation of the peptide into PLGA.[63] Recently, the applicability of PLGA-encapsulated SPf66 was investigated for oral delivery and the results showed that, in mice, an appropriate oral administration schedule could induce significant systemic antibody responses comparable to the conventional subcutaneous alum formulation.[61] The ability of PLGA-encapsulated SPf66 to elicit potent antibody responses was also demonstrated by the nasal route.[64] Although the development of SPf66 as a malaria vaccine has been discontinued due to its lack of efficacy in endemic populations,[65] these studies provide evidence that oral administration of antigen encapsulated in PLGA microparticles is capable of improving systemic immune responses compared with unencapsulated antigen.


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