Despite its clinical importance, a safe and effective RSV vaccine has not yet been developed. A number of factors contribute to the difficulty of this problem, including the early age of primary infection, the presence of maternal antibodies in the neonate, and the potential for vaccine-enhanced disease. To circumvent the problems associated with the inactivated viral vaccine, a number of laboratories have pursued the strategy of generating attenuated vaccine strains (13
), an approach that has been successful for many other viral pathogens. Unfortunately, separation of disease and immunogenicity has been problematic for RSV, perhaps because the wild-type strains of this virus are themselves so poorly immunogenic. Antibody responses, while protective of the lower airway, are slow to develop in immunocompetent children and do not prevent repeated infection of the upper airway (20
). Long lasting T-cell responses are induced by primary RSV infection (47
), but their protective efficacy is uncertain (3
), and their potential for mediating inflammatory pathology is well established (7
In this study we have focused on the poor immunogenicity of RSV and asked whether presentation of RSV antigens in the context of strong IFN-α/β induction would induce protective T-cell responses. This hypothesis was based upon the observation that, unlike the strongly immunogenic influenza virus, IFN-α/β is not detected in the serum or nasal secretions of RSV-infected children (25
). In addition to the well-known direct antiviral effects of IFN-α/β (1
), its role in shaping the adaptive immune responses to viral pathogens has also begun to be appreciated. IFN-α/β have been shown to promote antigen processing and presentation through the MHC class I pathway in DCs (30
), as well as the activation, expansion, and survival of CD8+
T cells (34
). Not surprisingly, most viruses have evolved mechanisms to limit IFN-α/β production and/or function (18
). In the case of RSV, the NS1 and NS2 proteins of both the human and the bovine strains antagonize IFN-α/β production by inhibiting IRF3 activation (4
). While it is not clear that antiinduction mechanisms encoded by RSV are more potent than those of other viral pathogens, the very low levels of IFN-α/β found in serum and nasal washings of RSV-infected patients (25
) suggest that this may be the case. Nonetheless, there appear to be multiple strategies employed by RSV to avoid immune recognition following primary and secondary infections, and one of these is the inability of this virus to activate the antigen-presenting DC compartment.
We have constructed a chimeric viral vaccine using a reverse genetic approach, inserting the RSV F protein gene into the backbone of NDV, another Paramyxoviridae
family member (44
). NDV, an avian virus that replicates poorly in mammalian cells or tissues due, at least in part, to its inability to prevent a strong IFN-α/β response in mammalian species, was chosen as a vector for these reasons (2
). While the details of IFN-α/β induction by NDV are not completely understood, the IFN-antagonizing functions of the viral V protein appear to be restricted to the avian host (49
). The RSV F gene was chosen as the target antigen for two reasons. Firstly, only antibodies directed at the F and G surface glycoproteins of RSV are associated with protection (11
), and secondly, of these two proteins, only F contains both CD4+
T-cell epitopes (58
). The G protein, which has been associated with the priming of Th2 responses, has no MHC class I-restricted peptide epitopes that are recognized by the BALB/c mouse (60
). We predicted that the NDV-F virus would be a much stronger inducer of IFN-α/β in BALB/c mice than RSV following i.n. instillation despite its inability to productively replicate in the mouse, and we found this to be true (Fig. ). We then looked to see whether NDV-F-immunized mice showed any correlates of protection upon challenge with RSV A2 and, if so, how this protection was mediated. Our objective in performing this experiment was to test the hypothesis that immunization with an IFN-α/β-inducing adjuvant would enhance the adaptive immune response to RSV. While mucosal NDV-F immunization with 5 × 105
PFU did not prevent infection, virus burden was decreased 10-fold without evidence of enhanced inflammation or Th2 cytokine production. RSV F-specific, CD8+
memory T cells were present in greater numbers in NDV-F-primed mice than in animals previously infected with RSV, suggesting that NDV-F may be a more effective immunogen than RSV itself.
The expectation that IFN-α/β induction would have an adjuvant effect was based on the ability of this cytokine to promote DC maturation as well as cross-priming during virus infection (37
). Our data show that the effects of both viruses on mDC cultures are largely IFN-α/β dependent; NDV is a much more effective activator but also induces much higher levels of IFN-α/β in the mDC population. Conversely, in Flt-3L-expanded cultures that contain pDCs as well, IFN-α/β levels and extent of maturation are equivalent. This is consistent with our hypothesis but does not explain the ability of NDV-F to protect IFNAR−/−
mice from RSV challenge. The in vivo study demonstrating that NDV-F is immunogenic even in the absence of the IFN-α/β pathway (Fig. ) suggests that NDV can activate DCs by alternate pathways as well. Evidence for this additional pathway is shown in Fig. , which illustrates maturation of IFNAR−/−
DCs, particularly by NDV, in BM-derived DCs cultured with Flt-3L. The mechanism underlying this IFN-α/β-independent pathway is beyond the scope of this present study, but we speculate that a population of cells present in the Flt-3L cultures is activated by virus to secrete an additional cytokine(s) that can also mediate DC maturation. Studies aimed at the characterization of these factors are currently in progress.
In addition to its inherently high adjuvant properties, as shown by our studies, NDV is an attractive vaccine viral vector in humans because of several additional characteristics. Importantly, NDV has proven to be safe in humans, as determined in several clinical trials focused on the use of this virus as an oncolytic agent in cancer patients (39
). In our experiments, we have used an attenuated viral strain of NDV (Hitchner B1) currently employed as a live attenuated vaccine in poultry against Newcastle disease. The lack of preexisting immunity against this virus in the vast majority of the human population represents another important factor that facilitates the use of NDV as a vaccine vector.
In conclusion, we have provided evidence that the RSV F protein is more immunogenic when presented by NDV-F than by RSV itself and that this correlates with an increased ability of NDV to activate antigen-presenting cells in vitro and to induce high levels of IFN-α/β in vivo. Mucosal NDV-F instillation can effectively prime a population of CD8+ memory T cells that persist and mount a strong IFN-γ response upon reintroduction of antigen. This vectored vaccine therefore offers a measure of protection against RSV challenge, while IFN-γ secreted by F-specific CD8+ T cells promotes a robust Th1 response to viral epitopes not yet seen by the host immune system. The mechanism of this adjuvant effect is largely mediated by the potent IFN induction by the avian viral vector. This approach to RSV immunization, one of expressing RSV antigens in vectors more capable of immune stimulation, may offer a useful alternative to the problems encountered with both live attenuated and inactivated vaccine preparations.