DNA encoding the attachment (G) or fusion (F) protein of respiratory syncytial virus induces protection in the absence of pulmonary inflammation

Gary P. Bembridge1, Nuria Rodriguez2, Regina Garcia-Beato2, Carolyn Nicolson3, Jose A. Melero2 and Geraldine Taylor1

Institute for Animal Health, Compton, Newbury, Berkshire RG20 7NN , UK1
Centro Nacional de Biologia Fundamental, Instituto de Salud Carlos III, Majadahonda, 28220 Madrid, Spain2
NIBSC, Blanche Lane, South Mimms, Potters Bar, Hertfordshire EN6 3QG, UK3

Author for correspondence: Gary Bembridge. Fax +44 1635 577263. e-mail Gary.Bembridge{at}bbsrc.ac.uk


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Significant protection against respiratory syncytial virus (RSV) infection was induced in mice vaccinated intramuscularly (i.m.) with DNA encoding the F or G protein of RSV. The amounts of IgG1 of IgG2a antibodies in mice immunized with DNA-G alone were similar. However, the antibody response in mice co-immunized with DNA-G and DNA encoding IL-4 (DNA-IL-4) was strongly biased towards IgG1. In contrast, the antibody response in mice co-immunized with DNA-G and DNA-IL-2, -IL-12 or-IFN-{gamma} was biased towards IgG2a. Mice vaccinated with DNA-F either alone or in combination with DNA encoding cytokines developed a predominant RSV-specific IgG2a response, which was most pronounced in mice co-immunized with DNA-F and DNA-IL-12 or -IFN-{gamma}. Vaccinated mice developed only a slightly enhanced pulmonary inflammatory response following RSV challenge. More significantly, and in contrast to mice scarified with recombinant vaccinia virus expressing the G protein, mice vaccinated i.m. with DNA-G did not develop pulmonary eosinophilia, even when the immune response was biased towards a Th2 response by co-administration of DNA-IL-4.


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Respiratory syncytial virus (RSV) is the single most common cause of viral bronchiolitis in young children and there is currently no effective vaccine. Indeed, vaccine development has proceeded with caution following the spectre of vaccine-augmented disease in recipients of a formalin-inactivated (FI)-RSV vaccine. Studies in mice suggest that this vaccine-augmented disease was associated with the induction of a Th2 response and a failure to induce protective antibody and cytotoxic T cells (Connors et al., 1992 , 1994 ; Graham et al., 1993 ).

DNA vaccination is an effective way of generating humoral and cell-mediated immune responses and has been shown to be effective against a range of pathogens, including RSV (Donnelly et al., 1997 ; Li et al., 1998 ; Schrijver et al., 1997 ; Tripp et al., 1999 ). However, studies on the effect of DNA vaccination on pulmonary pathology following RSV challenge have given conflicting results. Thus, although mice vaccinated intramuscularly (i.m.) with DNA encoding a soluble form of the F protein were protected against RSV infection, there was a significant increase in pulmonary cell infiltration, similar in magnitude to that seen in mice vaccinated i.m. with FI-RSV, although eosinophils were not detected (Li et al., 1998 ). In contrast, high levels of eosinophils were detected in bronchoalveolar lavage (BAL) from mice vaccinated i.m. with DNA encoding either the F or G protein and challenged with RSV (Tripp et al., 1999 ). However, the level of protection against RSV infection induced by DNA vaccination was not examined in the latter study.

Previous studies utilizing recombinant vaccinia viruses (rVV) in mice demonstrated that the F and G proteins of RSV prime different T cell responses and induce different patterns of pulmonary pathology following RSV challenge. Thus, vaccination of mice with VV-F primes Th1-like CD4+ T cells and CD8+ CTLs (Alwan & Openshaw, 1993 ; Alwan et al., 1993 ) and induces a peribronchiolar and perivascular infiltration of lymphocytes and polymorphonuclear leukocytes following RSV challenge (Stott et al., 1987 ). In contrast, VV-G primes Th2-like CD4+ T cells but no CTLs (Alwan & Openshaw, 1993 ; Openshaw et al., 1992 ) and induces a lymphocytic and eosinophilic pulmonary infiltration following RSV challenge (Openshaw et al., 1992 ). In the light of these studies with rVV and the discrepancies in those describing the ability of DNA encoding the F protein to augment pulmonary pathology, we investigated the effects of i.m vaccination with DNA-F or DNA-G on protection against RSV infection and on the development of pulmonary pathology.

DNA vaccines delivered i.m. in saline tend to induce strong Th1 responses in mice, which are reflected in high levels of IgG2a compared with IgG1 (Feltquate et al., 1997 ; Pertmer et al., 1996 ). In contrast, Th2 responses in mice are associated with high levels of IgG1 antibodies. Since Th2 responses to RSV in mice are associated with the induction of pulmonary eosinophilia following RSV challenge, we investigated the Th bias of the immune response induced in mice by i.m. vaccination with DNA-F and DNA-G. Furthermore, since the differentiation of T cells is influenced by the cytokine microenvironment at the time of activation, we also investigated whether the Th bias could be manipulated by co-administration of plasmids encoding cytokines. The consequences of any changes in Th bias on the development of pulmonary pathology following RSV challenge were also investigated.

The genes encoding the F protein from the Long strain of human RSV or murine cytokines (see Table 2) were cloned into the plasmid pI17 (a gift from J. Robertson, NIBSC, UK) under the control of the CMV immediate-early promoter. The G gene from the Long strain of RSV was cloned into plasmid pVR1012 (Vical). Endotoxin-free plasmid was produced by using the Qiagen Giga plasmid kit according to the manufacturer’s methods (Qiagen). Six-week-old, specific-pathogen-free, female BALB/c mice were purchased from Charles River Breeding Laboratories. The mice were immunized i.m. once, twice or three times at weekly intervals with 100 µg DNA encoding either the F or G protein, bilaterally, together with 100 µg empty plasmid or plasmid encoding cytokines. Three to 4 weeks after the final immunization, mice were challenged intranasally with RSV and lungs were removed 5 days later and assessed for virus load (Taylor et al., 1984 ).


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Table 2. Effect of co-immunization with DNA encoding cytokines on antibody responses and protection against RSV infection

 
Although a single immunization with pI17-F reduced virus replication in the lungs significantly compared with controls, only two of five mice were completely protected (Table 1). Two immunizations with pI17-F further reduced the level of virus replication and three immunizations resulted in complete protection in all five mice (Table 1). Similarly, a single immunization with pVR1012-G appeared to result in a reduced virus load, but this was not significantly different from control mice. The level of protection was increased in mice receiving three immunizations, although only two of five mice were completely protected. Of a total of 24 mice vaccinated three times with 100 µg DNA-F in the present study, 16 (66%) were completely protected against RSV infection. In contrast, only 2/24 (8%) mice vaccinated three times with DNA-G were completely protected. Li et al. (1998) reported that complete protection against RSV infection was only induced by two doses of 100 µg of a plasmid expressing a secreted form of the F protein and containing the rabbit {beta}-globulin intron II sequence upstream of the F gene. In order to determine whether the levels of protection induced by our current plasmids can be improved, we are currently investigating the ability of plasmids encoding secreted forms of the F or G proteins to protect against RSV infection.


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Table 1. Effect of weekly i.m. vaccinations on antibody responses and protection against RSV infection

 
As seen previously (Bembridge et al., 1998a ), mice scarified with VV-G or VV-F had 6- to 10-fold more cells in BAL than control mice, 5 days after RSV challenge (results not shown). In contrast, i.m. vaccination with DNA-F or -G did not result in substantial increases in the numbers of BAL cells when compared with relevant controls (Table 2). The largest increase observed in four replicate experiments was only 2·5-fold. Furthermore, analysis of cytocentrifuge preparations of BAL cells from DNA-vaccinated mice revealed that they were mostly lymphocytes, macrophages and neutrophils with few (often <1%) eosinophils, even in mice vaccinated with DNA-G (Fig. 1). In contrast, BAL from mice scarified with 2x106 p.f.u. VV-G contained 4–15% eosinophils 5 days after RSV challenge (Fig. 1). Flow cytometric analysis of BAL cells with MAbs to murine leukocyte differentiation antigens (Pharmingen) was performed as described previously (Bembridge et al., 1999 ). In contrast to VV-G-primed mice, CD8+ T cell numbers exceeded CD4+ T cells in BAL from all groups of DNA-vaccinated mice 5 days post-challenge (data not shown). These findings contrast with those of Tripp et al. (1999) , who reported high levels of pulmonary eosinophilia following RSV challenge in mice vaccinated i.m. with DNA-F or DNA-G. Our immunization regime used five times more DNA than that used by Tripp and colleagues. We are currently investigating whether the differences in pulmonary eosinophilia are due to differences in the dose of DNA. The finding that i.m. vaccination with DNA-G induced a significant reduction in pulmonary RSV titres but did not prime for pulmonary eosinophilia following RSV challenge is similar to our previous observations in mice vaccinated intraperitoneally with VV-G (Bembridge et al., 1998a ) and provides further confirmation that priming for pulmonary eosinophilia by the G protein is critically dependent upon the method of vaccination.



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Fig. 1. Polymorphonuclear granulocyte content in BAL from vaccinated mice. Mice were vaccinated i.m. with 100 µg plasmid DNA encoding the G protein either alone or in combination with 100 µg plasmid DNA encoding cytokines on three occasions, 1 week apart, or by scarification with 2x106 p.f.u. rVV expressing the G protein (VV-G) or {beta}-galactosidase (VV-{beta}-gal). Mice were challenged intranasally with RSV, 6–7 weeks after vaccination, and BAL cell samples were obtained 5 days after challenge and examined by light microscopy. The numbers of eosinophils ({bullet}) or neutrophils ({circ}) are expressed as percentages of the total number of cells counted.

 
The production of cytokines by RSV-stimulated splenocytes from vaccinated mice was determined as described previously (Bembridge et al., 1999 ). Mice vaccinated i.m. with DNA-F or DNA-G produced IFN-{gamma}, with little or no detectable IL-4 or IL-5. Thus, i.m. vaccination with DNA-F or DNA-G induced a strong Th1 cytokine response. This was reflected in the levels of IgG2a and IgG1 RSV-specific antibodies, analysed as described previously (Stott et al., 1987 ) in DNA-F-vaccinated mice, in which IgG2a antibodies predominated (IgG1:IgG2a of 0·1; Tables 1 and 2). However, levels of IgG1 and IgG2a antibody in mice vaccinated with DNA-G were equivalent (IgG1:IgG2a of 1; Tables 1 and 2).

In order to determine whether the Th1-biased response induced by i.m. vaccination with DNA could be manipulated by DNA encoding cytokines, we injected mice i.m. with mixtures of DNA-F or DNA-G and DNA encoding different cytokines. In the mouse, IL-4 can direct the development of Th cells into Th2 cells and increase IgG1 antibody responses (Swain et al., 1990 ), whereas IL-12 and IFN-{gamma} can induce the development of Th1 cells and increase IgG2a antibody responses (Seder & Paul, 1994 ). Previous studies have shown that treatment of mice with recombinant IL-12 can influence T cell priming by FI-RSV or VV-G, resulting in an increase in IgG2a antibody and a reduction in pulmonary eosinophilia (Hussell et al., 1997 ; Tang & Graham, 1995 ). However, attempts to bias the immune response to RSV using rVV expressing the F protein, together with murine cytokines, demonstrated that the effects observed were due mainly to the action of the cytokines on virus vector replication (Bembridge et al., 1998 b ). Since this is not a problem for DNA vaccination, mice were vaccinated i.m. with DNA-F or DNA-G and plasmids expressing IL-2, IL-4, IL-12 or IFN-{gamma}. Apart from mice vaccinated with DNA-F or -G and DNA-IFN-{gamma}, there were no significant differences in antibody titres at the time of challenge and 5 days after RSV infection (Table 1 and results not shown). Therefore, the effects of cytokine genes on antibody responses are shown only for mice 5 days after RSV challenge (Table 2). Vaccination with DNA-F or -G and DNA-IFN-{gamma} induced serum antibodies that were 3- to 5-fold lower pre-challenge compared with after RSV challenge. Vaccination with DNA-F and DNA encoding cytokines did not affect the overall IgG2a bias of the RSV-specific antibody response (Table 2). However, the IgG2a bias was most pronounced in mice vaccinated with DNA-F and DNA-IL-12 or DNA-IFN-{gamma} (Table 2). Whereas co-administration of DNA-F with DNA-IL-4 did not affect the isotype of the antibody response when compared with that induced by DNA-F alone, co-administration of DNA-G with DNA-IL-4 induced IgG1 antibody titres that were 8-fold greater than IgG2a titres. In contrast, IgG2a antibody titres were 3- to 10-fold greater than IgG1 titres in mice co-immunized with DNA-G and DNA-IL-2, -IL-12 or -IFN-{gamma} (Table 2). The apparent inability to switch the isotype response to the F protein with DNA-IL-4 is similar to that observed following immunization with rVV expressing F together with relatively low levels of murine cytokines (Bembridge et al., 1998b ). Only vaccination with rVV expressing F together with high levels of IL-4 resulted in the preferential induction of IgG1 antibody. It is possible that DNA encoding cytokines would be more effective if the F gene and the cytokine gene were expressed from the same plasmid as a fusion protein, as reported previously for ovalbumin and IFN-{gamma} (Lim et al., 1998 ). Nevertheless, DNAs encoding cytokines were able to bias the isotype of the antibody response induced by DNA-G, despite being expressed from different plasmids.

There was a significant reduction in lung virus titres in mice vaccinated with DNA-F or -G, with or without co-immunization with DNA encoding cytokines, when compared with controls vaccinated with a control plasmid (Table 2) or with DNA encoding the cytokine plasmid only (results not shown). However, DNA-IFN-{gamma} appeared to reduce the protective efficacy of both DNA-F and DNA-G, although this reduction was only significant for mice vaccinated with DNA-F and DNA-IFN-{gamma} (P<0·001). This reduction in protective efficacy may be related to a reduction in the serum antibody response in mice vaccinated with DNA-IFN-{gamma}. Thus, at the time of RSV challenge, antibody titres were lower in mice vaccinated with DNA-F and DNA-IFN-{gamma} (log10 3·3±0·1) or with DNA-G and DNA-IFN-{gamma} (log10 3·3±0·2) than in mice vaccinated with DNA-F (log10 4·0±0·3) or DNA-G (log10 3·9±0·3) alone. A similar reduction in antibody titres has been observed when DNA-IFN-{gamma} was co-injected with DNA encoding the rabies virus G protein (Xiang & Ertl, 1995 ).

Co-injection of cytokine DNA did not affect the pulmonary cellular response in either F- or G-primed mice. Thus, the BAL cell count in mice vaccinated with DNA-F or -G and DNA encoding cytokines was only 1·3- to 2·5-fold greater than that in control mice undergoing primary RSV infection. Despite the induction of a Th2-biased antibody response in mice immunized with DNA-G and DNA-IL-4, there were few eosinophils present in the BAL following RSV challenge (Fig. 1) and CD8+ T cells exceeded CD4+ T cells in BAL (data not shown) at 5 days post-challenge in all groups of mice. Furthermore, analysis of cytokines produced by RSV-stimulated splenocytes showed that there was a predominant production of IFN-{gamma}, with little or no detectable IL-4 or IL-5, in all groups of mice (data not shown), even in those vaccinated with DNA-G and DNA-IL-4. Thus, although DNA-IL-4 induced an IgG1-biased antibody response, the cytokine response in these mice was still predominantly Th1.

The ability to induce a protective immune response in BALB/c mice with DNA-F or -G without inducing an augmented pulmonary inflammatory response or pulmonary eosinophilia, following RSV challenge, reveals a promising strategy for producing an effective vaccine for use in humans. Furthermore, these studies highlight previous observations that induction of pulmonary eosinophilia in BALB/c mice by the G protein is critically dependent upon the method of vaccination. The present studies also demonstrated that co-injection of DNA encoding cytokines can influence the isotype of the antibody response, although the effects of the cytokines vary with the viral glycoprotein. However, the extent to which these findings are applicable to humans are not known, since the polarization of Th1 and Th2 responses in humans is less dramatic than in mice, and the effects of cytokines on human antibody subclass induction may differ. One major disadvantage of this type of immunization is the large quantity of DNA required to induce immunity, and further improvements in vector delivery, targetting of antigen-presenting cells and expression levels are probably required in order to make the immunization more efficacious. One current approach to these problems is the use of DNA-coated microparticles that can be fired directly into the epidermis, which requires far less DNA to induce immune responses (Feltquate et al., 1997 ; Pertmer et al., 1996 ). We are currently investigating the ability of this form of DNA immunization to induce protection against RSV infection and to protect against pulmonary pathology.


   Acknowledgments
 
This work was supported by the grants from the Ministry of Agriculture, Fisheries and Food, UK, the European Union #PL960637 and #98/1086 from the Fondo de Investigaciones Sanitarias. N.R. is the recipient of a pre-doctoral fellowship from the Instituto de Salud Carlos III.


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Received 6 March 2000; accepted 30 June 2000.