Venezuelan Equine Encephalitis Virus Replicon Particles Encoding Respiratory Syncytial Virus Surface Glycoproteins Induce Protective Mucosal Responses in Mice and Cotton Rats

Respiratory syncytial virus (RSV) is a major human pathogen that causes serious lower respiratory tract illness in infants and the elderly. Significant morbidity and mortality for RSV are especially common in certain high-risk pediatric populations such as premature infants and infants with congenital heart or lung disorders. RSV bronchiolitis in infants is associated with recurrent wheezing and asthma later in childhood (53, 76). There are currently no FDA-approved vaccines for prevention of RSV disease by active immunization. Immunoprophylaxis by passive transfer of a humanized murine RSV fusion (F) protein-specific antibody is licensed for much of the high-risk infant population but is not cost-effective in otherwise healthy infants, who represent the majority of those hospitalized with RSV. There is also a high rate of RSV reinfection during childhood, which suggests that a protective immune response to a vaccine may need to differ either quantitatively or qualitatively from that induced by natural infection. Previous attempts to develop RSV vaccines have faced significant obstacles. An experimental formalin-inactivated RSV vaccine in the 1960s induced exacerbated disease and death in some vaccinated children during subsequent natural infection. It was shown subsequently that the formalin-inactivated RSV vaccine induced serum antibodies with poor neutralizing activity in infants (50) and an atypical Th2-biased T-cell response associated with enhanced histopathology following experimental immunization in small animals (58, 68). Treatment of RSV antigens with formaldehyde modifies the protein with carbonyl groups, which preferentially induces Th2-type responses and leads to enhanced disease (47). Other attempts to generate RSV vaccines include using live-attenuated cold-adapted, temperature-sensitive mutant strains of RSV (10, 12-17, 22, 32, 39, 41, 42), protein subunit vaccines coupled with adjuvant (30, 56, 70, 73), and RSV proteins expressed from recombinant viral vectors, including vaccinia virus (52, 75), adenovirus (31), vesicular stomatitis virus (37), Semliki Forest virus (8), bovine/human parainfluenza virus type 3 (26), Sendai virus (64), and Newcastle disease virus (45). Although some of these vaccines showed promising preclinical data, no vaccine has been licensed for human use due to safety concerns and lack of efficacy data. RSV vaccines under development have not been tested in efficacy trials. In addition, many of these vaccines face significant hurdles when they are introduced into very young infants, who are one of the principal target populations for RSV vaccines. Infants have circulating maternal antibodies against RSV and against most of the candidate viral vectors, which likely would cause a blunting of the efficacies of these vaccines in vivo. The two surface glycoproteins of RSV, F protein and attachment (G) protein, are the major antigenic targets for neutralizing antibodies. Serum neutralizing antibodies in high titer are sufficient to protect the lower respiratory tract (9). F and G proteins, therefore, have been used separately or in combination in many experimental RSV vaccines. Immunization with purified F protein alone or F protein expressed from a recombinant viral vector such as vaccinia virus induces RSV-specific neutralizing antibodies, CD8+ cytotoxic T lymphocytes, and protection against subsequent RSV challenge in mice or cotton rats (52). Vaccination with G protein alone, however, often induces only partial protection against RSV challenge. In mice, the immune response against G is associated with eosinophilia and the induction of Th2-type CD4+ lymphocytes in some experiments (27, 35, 65). A key determinant for optimal vaccination against respiratory viruses, such as RSV, is the ability of the vaccine to induce mucosal immunity. This goal can be achieved by using a mucosal route for vaccination or possibly by use of a vaccine construct that preferentially induces mucosal responses. Protection in the upper respiratory tract has been demonstrated in several animal models (22, 51) and in humans (42) following immunization by the intranasal (i.n.) route and has been linked to the induction of virus-specific mucosal immunoglobulin A (IgA) antibodies. Venezuelan equine encephalitis (VEE) virus is an RNA virus of the Togaviridae family. Virus replicon particles (VRPs) are defective nonpropagating VEE particles developed by Pushko et al. in 1997 (60). VRPs have been used successfully and safely in immunization and challenge studies for a wide range of viral and bacterial pathogens in animal model systems (2, 4, 24, 28, 29, 36, 43, 59, 60, 63, 69, 71). More importantly, these particles induce mucosal immune responses after nonmucosal inoculation in animals (18, 28) and confer protection to the primary mucosal target tissue (25; E. M. Richmond, K. W. Brown, N. L. Davis, and R. E. Johnston, unpublished results). VEE virus is also known to be infectious by aerosol and intranasal (i.n.) routes, which would allow the VRPs to access target cells to induce an immune response (6, 7, 33). VRPs contain a modified positive-sense RNA viral genome designed to express the VEE nonstructural replicase proteins, but no VEE structural proteins, as the structural protein genes have been replaced by the gene encoding the heterologous antigen. These particles are produced in a cellular packaging system in which structural proteins are supplied in trans and only the modified viral genome is packaged into an intact VRP. The resulting replicons express high levels of antigens in infected cells and induce humoral and cellular immune responses in vivo (60). Moreover, these replicons are potential vaccine vectors for use in very young infants, since they display VEE viral coat proteins and thus are not neutralized by maternal RSV antibodies. Other advantages of using VRPs over other viral vaccines include the lack of preexisting immunity to VEE in the target populations and their systemic and mucosal adjuvant activities (67). Here, we tested whether VEE replicon vaccine candidates could induce effective mucosal protection against RSV following i.n. immunization in BALB/c mice or cotton rats. These two animal models had previously been shown to be semipermissive to RSV infection. BALB/c mice were used to delineate the underlying mechanism of vaccine-enhanced RSV disease, and cotton rats were used in preclinical testing for their ability to allow RSV replication to high titers. Combination of the results from these animal models allowed us to compare directly the immune responses induced by the vaccine to those induced by natural infection, both quantitatively and qualitatively, and to look at the ability of those responses to inhibit viral replication in both the upper and lower respiratory tracts. In this study, we found that VRPs encoding the RSV F protein induced both systemic and mucosal antibody responses. These VRPs also induced antigen-specific T cells in both the lungs and spleens of immunized animals. The T-cell response was Th1/Th2 balanced, and aggravated histopathology was not observed. In addition, following i.n. challenge of these animals with wild-type RSV, virus replication was below the level of detection. In contrast, animals vaccinated with VRPs encoding the RSV attachment protein G showed challenge virus replication in the upper but not the lower respiratory tract. These findings provide proof-of-principle that VEE VRPs expressing the RSV F protein can be used to prevent RSV infection.