DNA vaccination with both the haemagglutinin and fusion proteins but not the nucleocapsid protein protects against experimental measles virus infection

Bernd Schlereth1, Paul-Georg Germann,2, Volker ter Meulen1 and Stefan Niewiesk1

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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Plasmids that expressed the nucleocapsid, haemagglutinin and fusion proteins of measles virus (MV) were used to immunize cotton rats (Sigmodon hispidus) against intranasal MV infection. After immunization with all three plasmids, T cell responses and MV-specific antibodies were induced. A reduction in virus titre was observed in lung tissue from animals immunized with plasmids expressing the viral glycoproteins. Histologically, however, a moderate peribronchitis was observed after immunization with the plasmid expressing the fusion protein whereas, after immunization with plasmids expressing haemagglutinin or both glycoproteins, only mild or focal peribronchitis was seen. Immunization with the nucleocapsid did not reduce virus titres, probably because of the failure to induce neutralizing antibodies. A disadvantage of plasmid immunization was its inefficacy in the presence of MV-specific ‘maternal’ antibodies. This indicates that genetic immunization has to be improved to be a useful alternative vaccine against measles.


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Measles is still one of the most serious infectious diseases worldwide. Although a live-attenuated vaccine virus exists, roughly one million children die of measles virus (MV) infection every year, mostly in developing countries (Katz, 1995 ). Immunization at an early age is necessary in order to protect infants against infection. However, because of maternal antibodies, vaccine-induced seroconversion is often inhibited (Griffin, 1995 ). In addition, because of the immaturity of the neonatal immune system, early immunization in the absence of maternal antibodies results in inadequate levels of immune protection. As the WHO has set the eradication of MV as a public health goal, alternative vaccine candidates are needed (Wild, 1999 ).

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 (6–8 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 (1·5–2 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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Table 1. Immunization efficiency depends on the immunization dose, the use of stimulator DNA and the route of immunization

 
Using this immunization protocol, animals were immunized with pCG-H5, pCG-F1, pSC-N or pC-H5 and pCG-F1 combined. To test for MV-specific antibodies, diluted cotton rat serum (1:100) was bound to MV coated onto a 96-well plate and detected with a rabbit serum specific for cotton rat immunoglobulins (Virion Systems). Finally, a horseradish peroxidase-coupled goat serum specific for rabbit antibodies was used. To assay for neutralizing antibodies, twofold serum dilutions were incubated with 50 p.f.u. MV strain Edmonston for 1 h, plated in duplicate on Vero cells and checked for cytopathic effect on day 5. PCG-H5 induced high levels of MV-specific as well as neutralizing antibodies (Fig. 1a, b). PSC-N induced the same level of MV-specific antibodies as pCG-H5, but did not induce neutralizing antibodies. In comparison with pCG-H5, immunization with pCG-F1 induced lower levels of MV-specific as well as neutralizing antibodies (Fig. 1 a, b). The combined immunization with pCG-F1 and pCG-H5 induced the highest titres of MV-specific and neutralizing antibodies. Nine weeks after the first immunization, all animals were challenged by intranasal infection with 2x105 p.f.u. MV strain HU2. After 5 days, spleen cells were tested for MV-specific T cell proliferation and virus titres were determined and histological changes assessed from lung tissue.



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Fig. 1. Immunization efficiency of pSC-N, pCGH5, pCGF1 and pCGH5+pCGF1. (a)–(b) Groups of four cotton rats were immunized i.d. with pSC-N (d), pCGH5 (m), pCGF1 (r) and pCGH5+pCGF1 (j) twice with a 3 week interval. pcDNA3 (+) was used as a control. Three, 6 and 9 weeks after the first immunization, levels of MV-specific (a; by ELISA) and MV-neutralizing (b) antibodies were determined. (c)–(d) After 9 weeks, animals were challenged intranasally with 2x105 p.f.u. MV (HU2 strain). After 5 days, animals were sacrificed and MV-specific T cell proliferation in spleen cells (c) and the virus titre in lung tissue (d) were measured. The stimulation index of T cell proliferation of non-immune animals was 6. Reductions of virus titres in groups immunized with pCGH5, pCGF1 and pCGH5+pCGF1 were significant (P<0·0003; two-tailed paired t-test) in comparison with control DNA and pSC-N (indicated by asterisks), but did not differ significantly from each other.

 
To test for MV-specific T cell proliferation, 0·5 µg gradient-purified, UV-inactivated MV was coated (in 200 mM NaCO3 buffer, pH 9·6, at 4 °C) overnight onto 96-well plates. After two washes, spleen cells from immunized and non-immunized animals were plated onto MV-coated and untreated wells. Two days later, spleen cells were labelled with [3H]thymidine for 20 h and then harvested and measured as described previously (Niewiesk et al., 1997 ). Immunization with pSC-N and pCG-F1 induced good T cell proliferative responses, whereas the pCG-H5-specific T cell proliferation was low (Fig. 1 c). Five days after challenge, virus was titrated from the left lobe of the lung as described previously (Niewiesk et al., 1997 ). The three right lobes were fixed with PBS/5% formaldehyde and paraffin sections were cut and stained with haematoxylin/eosin for histological analysis. Immunization with pCG-F1 and the combined immunization with pCG-F1 and pCG-H5 resulted in strong reductions in virus titres, whereas immunization with pCG-H5 was not quite as efficient and immunization with pSC-N gave no protection at all (Fig. 1 d).

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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Fig. 2. Immunization with pCGH5 and pSC-N in the presence of MV-specific (‘maternal’) antibodies. (a)–(b) Groups of four cotton rats were immunized i.d. twice with a 3 week interval with pCGH5 (n, m) or pSC-N (s, d). In some groups (open symbols), 1 ml human serum was transferred 1 day before the first immunization by intraperitoneal injection. By using ELISA systems specific for human and cotton rat antibodies (a), the decline in passively transferred human antibodies (x) could be differentiated from actively generated cotton rat antibodies whereas, by neutralization assay (b), only the decline of neutralizing antibodies over time indicated human antibodies. (c) Nine weeks after the first immunization, animals were challenged with MV (HU2 strain) and virus titres in lung tissue were determined 5 days later. The difference in protection between groups immunized with pCGH5 in the absence or presence of ‘maternal’ antibodies was significant (indicated by asterisk; P<0·007; two-tailed paired t-test). Ser, Human serum.

 
Contradictory reports have been published about the ability of DNA immunization to overcome maternal antibodies (Hassett et al., 1997 ; Wang et al., 1998 ; Monteil et al., 1996 ; Le Potier et al., 1997 ). In piglets, genetic immunization with a plasmid expressing a viral glycoprotein of pseudorabies virus is not effective in the presence of maternal antibodies (Monteil et al., 1996 ; Le Potier et al., 1997 ). In contrast, immunization with a plasmid expressing a glycoprotein of rabies virus in a mouse model was reported to be protective in the presence of maternal antibodies (Wang et al., 1998 ). Also, a plasmid expressing the N protein of lymphocytic choriomeningitis virus induced CTL in mice and conferred some protection in the presence of maternal antibodies (Hassett et al., 1997 ). Here, in experimental MV infection, the induction of antibodies against both the external H glycoprotein and the internal N protein was inhibited in the presence of MV-specific antibodies and immunization with H protein conferred no protection in the presence of ‘maternal’ antibodies. In order to solve this problem, plasmid immunization may have to be improved by stimulating the immune system more potently by inclusion of cytokine genes or CpG motifs into immunizing plasmids or by better targetting of plasmids to antigen-presenting cells (Fennelly et al., 1999 ).

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.


   Acknowledgments
 
This work was supported in part by Deutsche Forschungsgemeinschaft and Bundesministerium für Bildung, Wissenschaft, Forschung und Technologie.


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Received 6 January 2000; accepted 2 February 2000.