Chimeric Plant Virus Particles Administered Nasally or Orally Induce Systemic and Mucosal Immune Responses in Mice |
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| Authors: | Frank R. Brennan Trevor Bellaby Sharon M. Helliwell Tim D. Jones S?ren Kamstrup Kristian Dalsgaard Jan-Ingmar Flock William D. O. Hamilton |
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| Affiliation: | Axis Genetics plc, Babraham, Cambridge CB2 4AZ, United Kingdom1.; Danish Veterinary Institute for Virus Research, Lindholm, DK-4771 Kalvehave, Denmark2.; and Department of Immunology, Microbiology, Pathology, and Infectious Diseases, Huddinge University Hospital, S-141 86 Huddinge, Sweden3. |
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| Abstract: | The humoral immune responses to the D2 peptide of fibronectin-binding protein B (FnBP) of Staphylococcus aureus, expressed on the plant virus cowpea mosaic virus (CPMV), were evaluated after mucosal delivery to mice. Intranasal immunization of these chimeric virus particles (CVPs), either alone or in the presence of ISCOM matrix, primed CPMV-specific T cells and generated high titers of CPMV- and FnBP-specific immunoglobulin G (IgG) in sera. Furthermore, CPMV- and FnBP-specific IgA and IgG could also be detected in the bronchial, intestinal, and vaginal lavage fluids, highlighting the ability of CVPs to generate antibody at distant mucosal sites. IgG2a and IgG2b were the dominant IgG subclasses in sera to both CPMV and FnBP, demonstrating a bias in the response toward the T helper 1 type. The sera completely inhibited the binding of human fibronectin to the S. aureus FnBP. Oral immunization of the CVPs also generated CPMV- and FnBP-specific serum IgG; however, these titers were significantly lower and more variable than those generated by the intranasal route, and FnBP-specific intestinal IgA was undetectable. Neither the ISCOM matrix nor cholera toxin enhanced these responses. These studies demonstrate for the first time that recombinant plant viruses have potential as mucosal vaccines without the requirement for adjuvant and that the nasal route is most effective for the delivery of these nonreplicating particles.Replicating vaccines such as live-attenuated bacterial (13) and virus (36, 40, 45) vaccines, as well as naked DNA vaccines (31), induce stronger and longer-lasting immune responses than conventional killed-subunit vaccines and also elicit protective cell-mediated immunity, often without the need for adjuvant. There are however, safety concerns over the use of these vaccines (24, 49), where persistence or reversion to virulence of the live vaccine strains and integration of the naked DNA vaccine into the host chromosome are of major concern. Recent technological advances, such as the use of more-effective adjuvants for both mucosal and systemic delivery (12, 16), liposome and ISCOM encapsulation of proteins and peptides (3, 19, 27), multiple antigenic peptides (35), and virus-like particles (VLPs) (1), have led to the development of more-effective subunit vaccines. To circumvent the safety concerns of replicating vaccines and to avoid the need for peptide synthesis and chemical coupling to a carrier such as keyhole limpet hemocyanin, we have been examining the utility of the plant virus cowpea mosaic virus (CPMV) as a carrier of peptides for immune recognition. CPMV is composed of 2 subunits, the small (S) and large (L) coat proteins, of which there are 60 copies of each per virus particle (46). Foreign peptides up to 37 amino acids in length can be expressed on either the L or S proteins; hence, 60 to 120 copies of a peptide can be displayed on a single virus particle (4b, 34). A peptide from the human immunodeficiency virus (HIV) gp41 glycoprotein is highly immunogenic when displayed on CPMV, eliciting high titers of HIV neutralizing antibodies (28, 29). Furthermore, a peptide derived from the VP2 protein of canine parvovirus (CPV) expressed on CPMV is immunogenic when administered to mink and subsequently protected the mink from a lethal challenge with the CPV-related mink enteritis virus (10).Most infectious viral and bacterial diseases involve colonization or invasion through mucosal surfaces by the pathogen, and hence it is important to develop vaccines that induce strong protective mucosal immune responses as a first line of defense. Where the organism, such as Vibrio cholerae and enterotoxigenic Escherichia coli, is restricted to the mucosa, strong mucosal immunity is often sufficient. However, when the organism disseminates from the mucosa into the bloodstream, a strong systemic response is also required to engender sterile immunity. Hence, the ideal mucosal vaccine should generate local immune responses at mucosal surfaces but also elicit generalized vaccine-specific immunity in the systemic lymphoid organs. The potential of CPMV-based vaccines for mucosal vaccination has not previously been determined.Oral immunization with particulate antigens, especially when presented as viable organisms, which can colonize the mucosa better than killed organisms, is effective at inducing local and generalized secretory and systemic immune responses (5, 43). However, the acidic pH and the presence of degradative enzymes in the gastrointestinal tract mean that when nonreplicating antigens are used, high concentrations are often required to elicit high levels of immunity (6). Another way to elicit mucosal immunity but circumvent the problems of oral immunization is to vaccinate via the intranasal route (2). Intranasal immunization requires up to 10-fold less immunogen for effective immunization and avoids the problems of low pH. Live vaccines (15, 37), virus-like particles (4, 25, 32), and synthetic peptides (17, 33, 44) in the absence of adjuvant have been shown to stimulate strong immunity when administered by this route. Furthermore, stimulation of the nasal mucosa, like stimulation of the intestinal mucosa, has been shown to be effective at generating protective immunity at distant mucosal sites (reviewed in reference 2).To assess the potential of CVPs as mucosal vaccines, mice were immunized intranasally or orally with CPMV expressing a peptide derived from the fibronectin-binding protein B (FnBP) D2 motif of Staphylococcus aureus (14, 42). The three fibronectin-binding domains, termed D1, D2, and D3, of FnBP have been shown to be immunogenic in mice and rats (7, 41). The CVPs were shown to be more immunogenic when administered (without adjuvant) via the intranasal route than when administered by the oral route, generating high titers of D2-specific antibody in serum and mucosa, and the serum antibody inhibited fibronectin binding to FnBP. |
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