In this study, we developed VEE replicon particles as vectors to deliver RSV surface glycoproteins and showed that when these vaccine candidates were delivered i.n., they induced immune responses comparable to, or greater than, those following wild-type virus infection.
VEE VRPs are attractive vaccine vectors for several reasons. First, they are less sensitive than most live viruses to type I interferons (74
), which allows enhanced protein expression in replicon-infected cells in the draining lymph nodes. Translation of gene inserts from other alphaviruses, such as Sindbis virus, could be inhibited by such interferons (61
). Second, parenteral or intradermal inoculation of VEE replicons induces mucosal responses directed toward the encoded antigens (28
), which are optimal for protecting against viruses at the respiratory mucosa. Although the mechanism underlying this unique mucosal immunogenicity of VRPs is not completely understood, protection and significant numbers of cells secreting antigen-specific IgA have been detected in the mucosa in immunized animals following VRP or VEE immunization via a nonmucosal route (18
). The study presented here focused on i.n. delivery as a first-step feasibility study with VRP vaccination for RSV. Other routes of nonmucosal delivery of VRP also were examined for the ability to generate immune responses and were found comparable.
Third, VRPs possess the ability to target specialized antigen-presenting cells such as Langerhans cells in the dermis and human monocyte-derived dendritic cells (DCs) (44
). Compared to VEE replicons, other alphavirus vectors are not as effective in infecting DCs. Sindbis virus does target DCs, but protein expression is shut down rapidly by the innate immune response (61
) and Semliki Forest virus does not infect DCs efficiently (32
). Activation of DCs would greatly enhance both the innate and adaptive immune responses to vaccine antigens.
Finally, when VRPs were coadministered with microbial antigens, they exhibit adjuvant activity in the systemic and mucosal immune compartments (67
). Although the mechanism of VRP-enhanced adjuvant activity is not well understood, the ability to enhance immune responses through adjuvant activity would likely play an important role in increasing vaccine efficacy in populations with immature immune systems, such as those of very young infants. Further study to develop RSV F protein vaccine with VRP as an adjuvant would be of interest.
Given the multiple advantages of VRPs over other viral vectors, we incorporated the genes for RSV fusion (F) and attachment (G) glycoproteins into the replicons and tested them in mice and cotton rats. F and G surface glycoproteins have been the targets for multiple experimental vaccines since these proteins are the targets for RSV neutralizing antibodies. Expression of RSV proteins from VRPs appeared authentic in every aspect. In BHK cells, VEE replicons expressed robust amounts of the encoded antigens. These antigens were expressed in a membrane-bound manner, which is consistent with published data on the distribution of F or G during RSV infection.
When inoculated i.n. in mice and cotton rats, VEE replicons induced RSV-specific binding and neutralizing antibodies in both the systemic and mucosal immune compartments. By inoculating VRPs via a mucosal site, we elicited a robust response against RSV in the respiratory tract and induced high levels of systemic RSV neutralizing antibodies. The RSV serum neutralizing titers induced by VRPs were directly proportional to vaccine dose, presumably due to an increase in antigen expression from higher numbers of VRPs. Remarkably, the serum neutralizing titers of VRP-RSV.F-vaccinated mice were higher than those following RSV infection, which demonstrates the potential of this vaccine. Similar serum neutralizing titers also were observed in mice vaccinated via the i.p. or i.m. routes. More importantly, mucosal IgA antibodies also were detected in the upper and lower respiratory tracts of i.n.-vaccinated animals. It should be noted that this comparison was performed with animals vaccinated with two doses of VRP-RSV.F versus animals vaccinated with a single dose of RSV. A single dose of RSV appeared to be equally effective in protecting animals from RSV challenge as two dose of VRP-RSV.F. In this study, we showed that vaccination with a single dose of VRP-RSV.F elicited a higher serum neutralizing titer at the 106-IU dose than RSV vaccination at 14 days postvaccination (Fig. ). The dosing and immunogenicity of RSV vaccines in mouse models and human infants are not perfectly correlated, so that it is difficult to extrapolate from our current data to say whether or not one or two doses would be immunogenic in young infants. Immunogenicity for human infants would have to be determined in clinical trials.
Possible combination of VRP-RSV.F vaccination with VRP-RSV.G may also broaden the immune response to RSV and give benefit to young human infants.
Another issue of importance is the presence of maternal antibodies in very young infants that could potentially suppress the immune response and efficacy to the VRP vaccine. Passively transferred antibodies have been shown to mediate suppression of the immunogenicity and efficacy of both replication-competent as well as defective vaccinia virus-based vaccines in rodents and nonhuman primates (20
). The effect of passively acquired RSV antibodies should be studied in future studies in VRP-vaccinated animals.
Although RSV-specific antibodies are shown to be effective in restricting viral replication during infection, cytotoxic T lymphocytes appeared to be required for resolution of infection and short-term protection against reinfection (11
). Both RSV-specific CD4+
T cells have been shown to confer protection to naïve animals against RSV challenge in adoptive transfer experiments (5
). Here, we demonstrated that vaccination with VRP encoding RSV F protein also induced F-specific CD8+
T lymphocytes. Upon stimulation with H-2Kd
MHC class I-restricted F epitopes, lung lymphocytes, or splenocytes from VRP-RSV.F-vaccinated mice secreted IFN-γ. In contrast, VRP-RSV.G replicons induced much lower humoral and cellular immune responses in comparison to those responses induced by VRP-RSV.F. This finding could be caused by several factors, such as a potential reduced expression level of G in vivo, the greater amount of glycosylation of G compared to F, and the need for complex processing of RSV G in vivo.
We used a homologous prime-boost strategy to evaluate the efficacy of VRPs in inducing neutralizing antibodies at various time points postimmunization. We found that a single prime boost was sufficient to induce a maximal level of neutralizing antibody responses. Further boosting with the same vectors had no significant effect on neutralizing titer, possibly due to the generation of antivector immunity.
When mice were challenged with RSV, only those that were vaccinated with VRP-RSV.F had viral replication reduced to undetectable levels in both the lungs and nasal turbinates. VRP-RSV.G-vaccinated mice that received a dose of 104
IU did not exhibit significant increases in neutralizing antibody titer, yet they were still protected in the lungs against RSV challenge. These mice may have produced low levels of neutralizing antibodies that could not be detected. In a semipermissive small animal model, such immune responses may be sufficient to restrict RSV in vivo; however, this level of immunogenicity is not likely to be effective in human subjects. RSV titers in the nasal turbinates of VRP-RSV.G-vaccinated mice remained high. This is consistent with the low levels of antibodies and lack of antigen-specific CD4+
T cells, which had been shown to correlate with upper respiratory tract protection in RSV-infected mice (55
This finding was supported by the real-time RT-PCR detection of relative quantity of RSV RNA following challenge in vaccinated animals. There was a greater reduction of RSV genome in the lungs of VRP-RSV.F-vaccinated or RSV-infected mice compared to those vaccinated with VRP-RSV.G, when using this sensitive detection approach.
One of the major hurdles to development of a RSV vaccine is concern over safety in RSV-naïve recipients. Increased mortality rates and exacerbated disease were seen in infants vaccinated with formalin-inactivated RSV in the 1960s during subsequent natural infection (38
). Enhanced histopathology with excessive cellular influx and skewed Th2-dominant cytokine production was seen in animals vaccinated with formalin-inactivated RSV following viral challenge (58
). In these animals, a highly disproportionate number of cells of the Th2 subset of CD4+
T cells was induced and found to be responsible for secreting IL-4 and IL-5, which in turn caused a pulmonary eosinophilic response (1
). We performed multiple experiments to determine the profile of the type of responses in VRP-vaccinated mice pre- and postchallenge. The subclass distribution of antigen-specific IgG was determined after immunization, to evaluate the balance of Th1 versus Th2 responses. Mice immunized with VRP-RSV.F showed a balanced IgG1/IgG2a ratio (~0.7) compared to RSV-infected STAT1-deficient mice genetically predisposed to Th2 responses upon RSV infection (~3.7). A skewed Th2/Th1 response predisposes animals to develop vaccine-enhanced RSV disease, as seen in FI-RSV-immunized animals. In addition, we evaluated lung histopathology and cytokine gene expression in VRP-vaccinated mice after live RSV challenge. There was no evidence of enhanced lung histopathology in VRP-vaccinated animals upon RSV challenge when compared to animals that were previously infected with RSV. Vaccinated animals had moderate alveolar, peribronchiolar, and perivascular infiltrates and no significant airway mucus production. Unvaccinated animals did show minor increases in lung inflammation with mild lymphocytic infiltration with a histopathology score slightly lower than that of the immunized groups, possibly due to the delay of the appearance of pathogenic responses seen in primary infection in naïve animals compared to vaccinated animals. In animals vaccinated with formalin-inactivated RSV, severe inflammation and cellular infiltration were seen with a significant increase in histopathology scores, as has previously been reported (58
Cytokine gene expression also was determined from lungs of these animals. Only IFN-γ gene expression was increased among all the cytokine genes tested. Infected groups had higher IFN-γ gene expression compared to uninfected controls. Interestingly, animals that had been vaccinated with VRP-RSV.F or VRP-RSV.G and those that were infected previously with RSV showed a dramatic increase in IFN-γ expression (~3 to 12 times greater depending on the groups) over groups that were not previously vaccinated or that were vaccinated with a heterologous VRP (VRP-MPV.F). This is probably due to the faster response times of T cells from vaccinated animals upon RSV challenge. This finding further suggests the development of properly balanced cellular immune responses in vaccinated animals upon RSV exposure.
In summary, we demonstrated that VEE VRPs encoding RSV F protein induced strong antigen-specific humoral and cellular responses on mucosal surfaces and protected animals against i.n. RSV challenge. This study provides strong feasible evidence for further development of this vaccine candidate for RSV.