1 Institute of Virology and Immunobiology, University of Wuerzburg, Versbacher Str. 7, 97078 Wuerzburg, Germany
2 Departments of Pathology, Yale University School of Medicine, 310 Cedar Street, New Haven, CT 06510, USA
Correspondence
Stefan Niewiesk
niewiesk{at}vim.uni-wuerzburg.de
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ABSTRACT |
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INTRODUCTION |
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In BALB/c mice, i.n. infection with wild-type VSV leads to infection of the brain via the olfactory nerve and fatal encephalitis (Reiss et al., 1998). In contrast, recombinant VSV, derived from an infectious clone, is attenuated and does not cause lethal encephalitis (Roberts et al., 1998
). Furthermore, a VSV recombinant expressing the haemagglutinin protein of influenza virus given i.n. protects mice against influenza virus challenge (Roberts et al., 1998
). However, residual pathogenicity (measured as weight loss) is observed after i.n. immunization of mice with VSV recombinants. The replication potential of VSV can be reduced by truncating the cytoplasmic domain of the envelope G protein. A mutant with a cytoplasmic tail of 29 aa truncated to 9 aa (CT9) grows in vitro as well as recombinant VSV, whereas a mutant with a truncation to 1 aa (CT1) is reduced 10- to 100-fold in virus production in vitro (Roberts et al., 1999
; Schnell et al., 1998
). In mice, immunization with VSV-HA (expressing the haemagglutinin of influenza virus A) results in weight loss but mice recover fully and are protected against influenza (Roberts et al., 1998
). A VSV CT9-HA recombinant causes less weight loss, mice recover fully and are protected against influenza (Roberts et al., 1998
). Both CT1-HA and CT9-HA induced immunity to influenza challenge (Roberts et al., 1999
).
In the present study, we tested the pathogenicity of wild-type VSV and the stepwise-attenuated recombinant VSVs expressing the MV haemagglutinin. We also investigated their protective capacity in the absence and presence of passively transferred MV-specific antibodies.
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METHODS |
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For challenge experiments, 5 days after i.n. infection with 5x105 p.f.u. MV strain HU2 in a volume of 50100 µl, animals were asphyxiated using CO2 and lungs were removed. Virus titres were determined as TCID50, as described (Niewiesk et al., 1997).
After i.n. or i.t. infection of cotton rats with recombinant VSV, animals were asphyxiated using CO2 at various time-points and lungs and brains were removed and weighed. Lung tissue was minced with scissors and both lung and brain dounced with a glass homogenizer. Serial 10-fold dilutions of virus-containing supernatant were assessed for the presence and levels of infectious virus in a 48-well microassay using BHK cells and CPE as an end-point. Plates were scored microscopically for CPE after 2 days. The amount of virus in inocula was expressed as the quantity of virus that could infect 50 % of inoculated tissue culture monolayers (TCID50). TCID50 was calculated according to Reed and Muench (Reed & Muench, 1938). The threshold of virus detectable was 102 TCID50.
Viruses.
Recombinant VSV and VSV-H (Schnell et al., 1996) were grown and titrated on BHK cells and MV strains Edmonston B and HU2 on Vero cells, according to standard procedures (Schnell et al., 1996
; Niewiesk et al., 1997
). To produce recombinant VSVs CT9-H and CT1-H, the MV haemagglutinin gene (strain Edmonston) was amplified by PCR using Vent polymerase (New England Biolabs). The forward primer was 5'-GGCCAATTACCGGTACAATGTCACCACAACGAGAC-3'. This primer contained an AgeI site (indicated in bold) for use in future cloning. The reverse primer was 5'-GGCCTTAAGCGGCCGCTATCTGCGATTGGTTCCATC-3'. This primer contained a NotI site (indicated in bold). The PCR product was digested with AgeI/NotI and ligated between the G and the L gene (similar to Roberts et al., 1999
) into the vectors pVSVCT-1 and pVSVCT-9 (Schnell et al., 1998
), which had been digested with XmaI/NotI. The plasmids obtained were called pVSVCT1-H and pVSVCT9-H. Virus was obtained from these infectious clones using published procedures and expression of MV-H was tested by Western blotting, as described (Roberts et al., 1998
). To concentrate CT1-H, virus preparations were pelleted in an ultracentrifuge for 1 h at 25 000 r.p.m. in a Beckman SW27 rotor through a 25 % sucrose cushion and resuspended in a smaller volume (100-fold).
ELISA.
For ELISA, 10 µg gradient-purified UV-inactivated MV ml-1 was coated in 200 mM NaCO3 buffer (pH 9·6) at 4 °C overnight, blocked with PBS/10 % FCS/0·05 % Tween 20 and incubated with diluted cotton rat serum (1 : 100) at 4 °C for 1 h. After washing, the plate was incubated with rabbit serum specific for cotton rat IgG (Virion Systems) for 1 h at room temperature. After washing, the plate was incubated with horseradish peroxidase-coupled goat serum specific for rabbit IgG (Zymed) for 45 min at room temperature and was subsequently developed with 0·5 mg o-phenylenediamine ml-1 in buffer (35 mM citrate/66 mM Na2HPO4, pH 5·2) and 0·01 % H2O2. The plate was read at a wavelength of 490 nm against a reference reading at 405 nm.
Neutralization tests.
Serum 2-fold dilutions were incubated with 50 p.f.u. MV strain Edmonston for 1 h at 37 °C and plated in duplicate onto 104 Vero cells per well of a 96-well plate. At 5 days later, infection of wells (50 %) was determined microscopically. The titre was defined as the reciprocal of the last protective serum dilution, as calculated from duplicate measurements.
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RESULTS |
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To test the immunogenicity and protective capacity of CT9-H and CT1-H, naive cotton rats were immunized i.n. with 5x105 and 2x107 p.f.u. I.n. immunization of seronegative animals with 2x107 p.f.u. CT9-H and CT1-H resulted in titres of MV-neutralizing antibodies and reduction in virus titres in lung tissue (Table 1) comparable to those after immunization with VSV-H (Schlereth et al., 2000b
). As with VSV-H, CT9-H induced neutralizing antibodies in the presence of MV-specific antibodies and protection against challenge after i.n. immunization (Table 1
). In contrast, i.n. immunization with CT1-H in the presence of MV-specific antibodies resulted in lower titres of MV-neutralizing antibodies (P<0·0001 compared to CT9-H). Immunization of seronegative animals with 5x105 p.f.u. CT9-H and CT1-H was clearly different in efficiency. The induction of MV-neutralizing antibodies by CT9-H (NT titre 610±300) and protection against challenge were comparable to immunization with the high-dose inoculum (2x107 p.f.u.). In contrast, after immunization with 5x105 p.f.u. CT1-H, the titre of neutralizing antibodies was clearly reduced (NT titre 130±90), as was protection (P<0·02 compared to CT9-H). I.n. immunization with 5x105 p.f.u. CT1-H in the presence of MV-specific antibodies did not induce MV-specific or -neutralizing antibodies, nor did it induce protection against i.n. challenge with MV (Table 1
and Fig. 4
). In contrast, immunization with 5x105 p.f.u. CT9-H induced MV-specific and -neutralizing antibodies and protection against i.n. challenge.
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DISCUSSION |
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So far, neutralizing antibodies are the only proven entity of the immune system to protect against measles, although it has always been assumed that T cell responses play a part because patients with defects in the T cell response have not been able to clear the virus (reviewed by van Els & Nanan, 2002). However, in cotton rats, the induction of a strong CD4 T cell response against the nucleocapsid by plasmid immunization did not lead to protection (Schlereth et al., 2000a
). Immunization in the presence of MV-specific antibodies inhibits completely the generation of neutralizing antibodies in cotton rats (Schlereth et al., 2000b
) and strongly reduces the CD4 T cell response. Although reduced, the T cell response is still present but is not protective against i.n. infection with MV (unpublished results). For this reason, we have concentrated our efforts on a vector system known to induce high levels of neutralizing antibodies.
In humans, the attenuated vaccine virus does not induce protection in the presence of maternal antibodies (Katz, 1995). To overcome this problem, MV proteins have been expressed in vector systems derived from attenuated viruses (Durbin et al., 2000
; Wyde et al., 2000
; Stittelaar et al., 2000
; Weidinger et al., 2001
) and plasmids (Schlereth et al., 2000a
; Polack et al., 2000
). In experimental models, like monkeys and cotton rats, these vectors induced protective immunity in seronegative animals. In the presence of maternal antibodies, however, they were either not tested or failed to induce a good immune response after single immunization (Stittelaar et al., 2000
; Weidinger et al., 2001
; Schlereth et al., 2000a
). The only system in our hands to overcome this problem was a recombinant VSV expressing MV-H as a passenger protein (Schlereth et al., 2000b
). In seronegative animals, a good immune response was induced by live virus. In contrast, UV-inactivated virus was not able to induce an immune response at all. Although live VSV-H is able to induce active immunity in the presence of MV-specific antibodies, the generation of neutralizing antibodies is reduced 10-fold. In addition, the route of immunization is important. I.n. immunization, but not i.p. (Schlereth et al., 2000a
) or oral immunization, in the presence of MV-specific antibodies (unpublished results) led to protection against measles.
Because VSV-H at high titres was still able to infect the brain and induce weight loss, the contribution of brain versus lung infection in inducing immunity in the presence of MV-specific antibodies was evaluated. After i.c. inoculation of small volumes of virus, no immune response was observed. This is in line with previous observations that inoculation of virus into the draining cerebrospinal fluid induces immune responses, whereas inoculation into brain parenchyma does not (Stevenson et al., 1997). In contrast, i.t. immunization induced immune responses comparable to i.n. immunization. Why replication of VSV in lung tissue is able to induce a reduced (in comparison to naive animals) but protective immune response in the presence of MV-specific antibodies is not clear. It is possible that the lung is a very conducive environment in that the virus here is able to target dendritic cells at a high frequency and that local replication induces a number of immune stimulatory molecules (e.g. interferons). In addition, there might be an advantage for the virus to be relatively inaccessible to serum IgG. The delayed appearance of neutralizing antibodies in the circulation might also be explained by the induction of the immune response at the respiratory mucosa.
As immunization via the respiratory mucosa was important and this may lead to infection of the brain via the olfactory nerve, VSV-H was attenuated further by molecular means. It has been shown that the G protein of VSV directs budding (and thereby replication) efficiency of the virus. There do not appear to be specific interactions of the cytoplasmic tail in this process but mutants with a truncated cytoplasmic tail (9 or 1 aa instead of 29 aa) produce fewer virus particles per infected cell (Schnell et al., 1998). CT9-H never induced weight loss or clinical signs in cotton rats. After inoculation at high titre, CT9-H grew in brain tissue but was not found in the brain after low-dose inoculation. For cotton rats, CT1-H was not found in the brain but was slightly overattenuated in that it did not induce as good an immunity at low doses in seronegative animals as CT9-H. Immunization in the presence of MV-specific antibodies always leads to a strong reduction in immune responses (Schlereth et al., 2000b
). In the presence of MV-specific antibody, a low dose of CT1-H was not able to induce immunity to MV but a higher dose did yield partial protection. Thus, CT1-H is overattenuated as a MV vaccine vector when used in the presence of MV-specific antibodies. However, this problem can be overcome, at least partially, by increasing the virus dose.
VSV has not only been used to efficiently express MV-H but also for a variety of different viral proteins that proved to be immunogenic in rodents. In addition, VSVs expressing proteins of simian immunodeficiency virus (SIV) and human immunodeficiency virus (HIV) have been successful in protecting rhesus macaques against AIDS after challenge with the highly pathogenic SIV/HIV chimera, SHIV 89.6P (Rose et al., 2001). There has been no evidence in this model for brain infection in adult monkeys after i.n. inoculation of VSV vectors.
Based on our results in the cotton rat model, we feel that studies in a non-human primate model for measles (such as the infant rhesus macaque model) (Zhu et al., 2000) should be undertaken with VSV-H and the more attenuated CT1-H and CT9-H mutants as well. If these studies show protection in the presence of maternal antibody to measles, the vector system should be moved to human clinical trials.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 18 December 2002;
accepted 28 April 2003.
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