In the RSV genome, the G protein is an envelope glycoprotein that is the most important and vulnerable to antigenic variation. Thus, RSV was divided into two subtypes, A and B, according to the antigenic and genetic differences, mainly within the G protein
. The predominant subtype of RSV belongs to type-A, which prevails in most areas of China; the A-subtype of the virus is also more pathogenic
. The A2 and Long strains are two “standard” RSV-A subtypes. In this study, the Long strain of RSV was chosen for reverse genetics research.
To date, several examples have been reported regarding the successful construction of full-length infectious cDNA clones based on RSV-A2, X, and Long strains
. RSV-A2 was originally isolated in the 1960s, while RSV-X is a clinical isolate strain, 98-25147-X, isolated in 1998; the RSV Long strain is a strain of VR-26D from the American Type Culture Collection (ATCC). Previous research focused mainly on the RSV-A2 strain, including its different mutant forms, such as the deletions of G, SH, NS1, and NS2 genes, which have been studied extensively in vivo and in vitro
. Reverse genetics systems for RSV-X, a clinical RSV isolate, and RSV-X-ΔG were also reported
. The principles and methods for the construction and recovery of these strains are almost the same as those for the laboratory BRSV isolate strain A51908, described by Yunus
, including plasmids encoding the full-length viral antigenome and plasmids containing the viral structural protein genes N, P, L, and M2-1. Then, the resulting five plasmids were transfected into sensitive cells with the assistance of T7 RNP for the purpose of obtaining infectious recovered viruses. Lo et al. reported that rLong was rescued in BHK-SR19 cell lines using the Sindbis virus expressing T7 RNA polymerase
. However, no follow-up studies were reported on this recombinant virus. Our work represents another Long virus reverse genetics system, which is similar to that of Lo et al., except for the source of the T7 RNP (a recombinant vaccinia vTF7-3) and the cell lines used for virus recovery. Beyond establishment of the reverse genetics system, further studies on viral passage and an immunogenicity study of the recovered Long virus are reported in this paper.
Replication of the recovered virus showed that titers of rLong were ~100-fold lower in vitro than the parental strain, and that the replication of rLong-ΔG virus declined slightly compared with rLong. These results were consistent with the previous work on RSV-A2 and RSV-X reverse genetics systems, with recovery in Vero, 293 T, and A549 cells
. We hypothesized that these reduced recombinant viral titers in vitro may be as a result of the efficiency limits with recombinant plasmids transfection and virus rescue system. Replication of rRSV-X-ΔG in the cotton rat is highly attenuated. Indeed, the ΔG virus had no detectable viral load in the lungs of cotton rats on day 3
. This was consistent with the work from the Collins lab at the National Institutes of Health (NIH) where the ΔG was generated in the background of strain A2
, but the ΔG viruses for both the A2 and X strains were considered to be over-attenuated as vaccine candidates. Unexpectedly, we found that the virulence of our rLong-ΔG strain was less than 1 log attenuated in the lung of Balb/c mice on day 3, indicating that the rLong-ΔG virus was not over-attenuated. Thus, this newly constructed ΔG virus may be less attenuated than previously described ΔG mutants. That is, the Long strain may be less dependent on G for in vivo replication than the X or Long strain and may therefore be more suitable for attenuated vaccine strain screening. This is an important finding of our study on the recombinant ΔG Long virus. G protein is not required for infection in vitro but it may be an important stimulus for efficient RSV growth in vivo
[19, 27], and the Long-ΔG virus may be useful for finding the appropriate level of attenuation in recipients without sacrificing immunogenicity and ensuring a less attenuated phenotype. Indeed, an rRSV strain with the G protein deleted was rescued successfully
. However, the complete absence of the G protein severely restricted replication in vivo and thus limited the potential of the rRSV as a vaccine, as seen with the A2 and X strains
[19, 29]. Although the G protein is not an essential ingredient for viral replication in vitro, the viral titer and immunogenicity of the ΔG mutant could be affected. However, in this study, although viral virulence of recombinant ΔG long viruses was diminished, high titers of neutralizing antibodies for our rLong-ΔG virus in mice were induced and the protective antibody titers persisted for up to 21 days post-challenge with no significant decline. Furthermore, the protective efficacy of the rLong virus in lung tissue was up to 100%. In contrast, residual virus could be still detected in the NTL, which also suggested that the protective efficacy of our recombinant virus in the lower respiratory tract was better than in the nose. Alternatively, we also considered that the F protein is one of the major stimuli for virus neutralizing antibodies, which may be why titers of neutralizing antibodies did not decrease significantly with rLong-ΔG-EGFP.
For RSV vaccine candidates, the CD8+ T cell response has special significance because the CD8+ T cell response is not only directly involved in clearing virus and virus-infected cells but also potentially regulating CD4+ T cells at different stages of differentiation toward activating and prompting CD4+ T cell differentiation in a Th1-direction and producing Th1-type cytokines (IL-2, IL-12, IFN-γ)
[30–32]. Although the mechanism by which CD8+ T cells mediate the inhibition of RSV vaccine-enhanced pulmonary eosinophilia is currently unknown, CD8+ cytotoxic C lymphocytes (CTLs) do appear to play an important role in regulating the immune response through the secretion of cytokines, especially IFN-γ
. Previous work by several laboratories has shown that F- or M2-specific CD8+ T cell responses inhibited RSV vaccine-enhanced pulmonary eosinophilia because the production of IFN-γ can inhibit Th2-CD4+ T cell development in vitro
. Here, the number of peripheral blood mononuclear cells (PBMCs) secreting IFN-γ and IL-2 in response to RSV antigen indicated that both rLong and its G-replacement mutant were confirmed to produce CD8+ T cell immune responses due to the secretion of IFN-γ and IL-2. Bukreyev reported that RSV glycoprotein, through the cysteine-rich region of G (“GCRR”), a segment conserved in wild-type isolates worldwide, enhances the generation of an effective CTL response against RSV in BALB/c mice
. Our rLong virus also stimulated a higher level of IFN-γ in comparison with its G protein deletion form. We considered that one possible reason may be that the recombinant entire Long virus contains the GCRR, while the sequence is absent in the ΔG mutant strain.
Another significant consideration for any RSV vaccine is the balance of the immune response. Effective vaccines for RSV must elicit balanced T cell responses. Immune responses dominated by type-2 T cells against RSV antigens are believed to cause exaggerated respiratory tract disease and may also contribute to inflammation
. Findings from several laboratories have established that immunization with highly purified native or vaccinia virus-expressed recombinant G protein primed naive BALB/c mice for pulmonary eosinophilia upon subsequent challenge with infectious RSV
[36–38]. In contrast, Elliott reported that the immunization of BALB/C mice with rA2cpΔG177-220 or rA2cpΔG150-222 was immunogenic, and pulmonary eosinophilia was diminished significantly
. Thus, the tendency of the rA2 strain with a G protein mutation to elicit dominant type-2 responses was lessened significantly with no apparent loss of immunogenicity. In this study, serum IgG1 and IgG2a isotypes profiles indicate a Th2- and Th1-type-biased response, respectively; the levels were determined and the corresponding IgG1/IgG2a ratio was calculated to evaluate the CD4+ T response. Regarding the RSV Long strain, rLong and rLong-ΔG-EGFP also resulted in predominantly Th1-type CD4+ T cell responses, shown the lower ratio of IgG1/IgG2a, which was completely different from that with FI-RSV. Also, levels of the IgG2a antibody related to a Th1-CD4+ T response in rLong-ΔG-EGFP-immunized mice was higher than its counterparts. The region of amino acids 149–200 of the attachment (G) glycoprotein may be responsible for type-2 T cell responses and pulmonary eosinophilia
. This indicates that a Th2 T cell response, which is associated with pulmonary eosinophilia, could be diminished with our ΔG Long strain.
In addition to possessing strong immunogenicity, rLong-ΔG-EGFP performed better from the perspective of a balanced Th1/Th2 response. However, other research has shown that the RSV G glycoprotein is not necessary for vaccine-enhanced disease induced by immunization with FI-RSV
[40, 41]. RSV vaccine-enhanced disease is not due to antigenic content, but rather concerns the pathway of antigen processing, and thus G should not necessarily be excluded from potential vaccine products
. Polack also reported that the GCRR of the G glycoprotein is a potent inhibitor of inflammatory cytokine production. By inhibiting Toll-like receptor 4 (TLR4) activation and NF-κB nuclear translocation, it antagonizes the pro-inflammatory effect of the F protein regulating the innate immune response, indicating that it has broad anti-inflammatory properties; therefore, the production of inflammatory cytokines responsible for pulmonary eosinophilia could be prevented
. The role of G protein in inducing vaccine-enhanced disease or whether G should be excluded from RSV vaccine components needs to be clarified. Given that the mechanism(s) of RSV vaccine-enhanced disease is not understood, further exploratory efforts for RSV vaccines are worthwhile. And also, For RSV vaccine candidate development, high levels of neutralizing antibodies, CD8+ T cell responses, and a balanced T cell response should be taken into consideration. This study provides a platform for reverse genetics manipulation in discovering “ideal” attenuated RSV candidates and also allows examining the contribution of each viral component to vaccine-enhanced disease.