Institute of Virology and Immunobiology, University of Würzburg, Versbacher Str. 7, 97078 Würzburg, Germany1
Novartis Pharma AG, Preclinical Safety, WS-2881.4.07, CH-4002 Basel, Switzerland2
Author for correspondence: Stefan Niewiesk. Fax +49 931 201 3934. e-mail niewiesk{at}vim.uni-wuerzburg.de
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Abstract |
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DNA immunization has been suggested as an alternative to a live vaccine because plasmids are not replication-competent and, after in vivo transfection, virus protein is produced within dendritic cells and presented towards the immune system in its authentic form (for review see Koprowski & Weiner, 1998 ). It was shown that plasmids expressing the nucleocapsid (N), fusion (F) and haemagglutinin (H) proteins of MV are immunogenic in mice and rabbits (Cardoso et al., 1998
; Fooks et al., 1996
; Yang et al., 1997
). In addition, mice immunized neonatally with a plasmid expressing the H protein developed a good Th1-type response (Martinez et al., 1997
), which is thought to be beneficial for overcoming measles. As mice are not susceptible to intranasal infection with MV, these authors did not test whether DNA immunization induced protective immunity.
In contrast to mice, cotton rats (Sigmodon hispidus) can be infected intranasally with MV and live virus can be isolated from lung tissue, draining lymph nodes and, to a lesser degree, from spleen cells (Wyde et al., 1992 , 1999
; Niewiesk et al., 1997
). In this model system, we have tested DNA immunization with plasmids expressing the F (pCG-F1), H (pCG-H5) and N (pSC-N) proteins of MV (kindly provided by R. Cattaneo and M. Billeter, Zurich) for their immunogenicity and protective capacity. These plasmids were chosen because they express the respective MV proteins well in transfected cells in tissue culture (Schlender et al., 1996
; Huber et al., 1991
).
In order to establish a good immunization protocol, pCG-H5 was selected because the H protein is an important target in humans for T cell and neutralizing antibody responses (Griffin, 1995 ). For intramuscular (i.m.) immunization, the gluteal muscles of cotton rats (68 weeks old) were injected bilaterally with plasmid DNA (1 µg/µl in PBS) after treatment with cardiotoxin (Latoxan) to increase DNA uptake and enhance immune responses. For intradermal (i.d.) immunization, plasmid DNA (1·52 µg/µl in PBS) was injected into the lateral flank at two different sites. Previous work has suggested that i.d. plasmid immunization is superior to i.m. immunization (Boyle et al., 1997
) and that the addition of bacterial DNA might increase the efficiency of immunization due to immune-stimulatory sequences (CpG motifs) (Krieg et al., 1998
). We have chosen the plasmid pcDNA3 (Invitrogen), which contains six immune-stimulatory sequences, for co-immunization with pCG-H5. As shown in Table 1
, the immunization efficiency increased with the amount of plasmid used. The addition of co-stimulatory DNA enhanced antibody production and, after i.d. immunization, antibodies were induced earlier and to higher titres than after i.m. immunization. The optimal immunization schedule was i.d. immunization twice with a 3 week interval with 150 µg pCG-H5 and 50 µg pcDNA3.
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Immunization with vectors expressing the N protein in the rat and mouse model of MV encephalitis (Bankamp et al., 1991 ; Fennelly et al., 1995
; Fooks et al., 1995
) led to protection against CNS infection. CD4+ T cells alone are sufficient to protect against encephalitis and no neutralizing antibodies are required (Finke & Liebert, 1994
). In contrast, resolution of lung infection in cotton rats seems to require neutralizing antibodies.
Similar to our data, immunization with a mycobacterium expressing the N protein did not protect monkeys against MV infection, although it ameliorated the histological changes seen in lung tissue in comparison with control animals (Zhu et al., 1997 ). In cattle, a vaccinia virus expressing the N protein of rinderpest virus (a close relative of MV) was protective against challenge with a virus strain of low virulence, but not against a highly virulent strain (Ohishi et al., 1999
). In contrast, a vaccinia virus expressing the H protein of rinderpest virus protected against challenge with the highly virulent strain (Yamanouchi et al., 1993
). However, immunization of monkeys with immune-stimulatory complexes (ISCOM) containing the H and F proteins in the presence of maternal antibodies failed to induce neutralizing antibodies (van Binnendijk et al., 1997
), but T cell responses were induced and were sufficient to protect monkeys against challenge. Clinically, it was observed that patients with a defect in the humoral immune response were able to resolve MV infection (Bruton, 1953
; Good & Zak, 1956
), whereas patients with a T cell defect did not (Nahmias et al., 1967
).
For histological analysis of cotton rats immunized with pCG-H5 and pCG-F1, slides were coded and evaluated in a blinded fashion. The lesions were described according to their distribution and graded semi-quantitatively in their severity with a scale of 0 (no abnormalities detectable), 1 (mild, histopathological changes in at least one lobe), 2 (moderate, lesions in at least two lobes) and 3 (severe, lesions affecting two or more lobes). After decoding, the mean severity of findings in a group was calculated. After simultaneous immunization with both pCG-F1 and pCG-H5, minimal focal peribronchitis (grade 1) with alveolar histiocytosis and leucostasis was seen. Mild peribronchitis was seen in animals immunized with pCG-H5 (grade 1). Immunization with pCG-F1 led to moderate peribronchitis (grade 2) and a more severe histiocytosis and lymphocytic infiltration (grade 2). In addition, two animals showed alveolitis and one showed focal eosinophilic infiltrations. It is interesting to note that, as judged by histology, H was superior to the F protein. The combination of both was better than single immunization with either protein if neutralizing antibodies, T cell responses, virus titre and histology were analysed in combination.
One obstacle in immunizing infants against measles is the inhibition of vaccine-induced seroconversion by maternal antibodies. In order to mimic maternal MV-specific antibodies, a human serum was used. One ml of this human serum was standardized by using the human anti-measles serum (2nd International Standard 1990, 5 IU/ml; National Institute for Biological Standards and Control, Potters Bar, UK) and contains 16 IU (titre of 320 by neutralization test, titre of 256 by haemagglutination-inhibition assay). The serum contains antibodies specific for MV N, F and H proteins (data not shown). After transfer of 1 ml of this human serum into cotton rats, seroconversion after immunization with MV was blocked effectively (data not shown). Because of the use of heterologous serum, a differentiation between human antibodies from cotton rat antibodies is possible by ELISA. Thus, passively transferred maternal antibodies are clearly distinguishable from antibodies induced by immunization. After transfer of 1 ml human serum into cotton rats, MV-specific human antibodies were detectable by ELISA for 6 weeks (Fig. 2 a). With this system, the efficiency of immunization with pSC-N or pCG-H5 in the presence of passively transferred antibodies was tested. One day and again 3 weeks after serum transfer, cotton rats were immunized with pSC-N or pCG-H5. In the presence of human serum, the generation of MV-specific antibodies after immunization with pCG-H5 and pSC-N was severely reduced (Fig. 2 a
) and the generation of neutralizing antibodies after immunization with pCG-H5 was completely eliminated (Fig. 2 b
). In consequence, no protection against virus challenge was achieved after immunization with pCG-H5 in the presence of serum (Fig. 2 c
). In addition to the lack of protective immunity after immunization with pCG-H5, a severe peribronchitis (grade 3) and mild diffuse histiocytosis and lymphocytic infiltration in lung tissue were seen.
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In summary, good protective immunity against MV infection was achieved by immunization with plasmids expressing the H and F proteins of MV, but only in the absence of MV-specific maternal antibodies.
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Acknowledgments |
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References |
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Received 6 January 2000;
accepted 2 February 2000.