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Live Measles Vaccine Expressing the Secreted Form of the West Nile Virus Envelope Glycoprotein Protects against West Nile Virus Encephalitis

  1. Philippe Desprès1,
  2. Chantal Combredet2,
  3. Marie-Pascale Frenkiel1,
  4. Clarisse Lorin2,
  5. Michel Brahic2 and
  6. Frédéric Tangy2
  1. 1 Unité des Interactions Moléculaires Flavivirus-Hôtes Paris, France
  2. 2 Unité des Virus Lents, Institut Pasteur, Paris, France
  1. Reprints or correspondence: Dr. Frédéric Tangy, Unité des Virus Lents, Institut Pasteur, 28 rue du Dr Roux, 75015 Paris, France (ftangy{at}pasteur.fr) or Dr. Philippe Desprès, Unité des Interactions Moléculaires Flavivirus-Hôtes, 25 rue du Dr Roux, 75015 Paris, France (pdespres{at}pasteur.fr).
 
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Abstract

The Schwarz strain of measles virus (MV), a live attenuated RNA virus, is one of the safest and most effective human vaccines available. Immunization with MV vaccine expressing heterologous antigen is an attractive strategy to prevent emerging viral diseases. West Nile virus (WNV), which recently emerged in North America, is an important mosquito-borne flavivirus that causes numerous cases of human encephalitis, thus urging the development of a vaccine. To evaluate the efficacy of recombinant MV for the prevention of WNV encephalitis, we constructed a live attenuated Schwarz MV (MVSchw-sEWNV) expressing the secreted form of the envelope glycoprotein from the virulent IS-98-ST1 strain of WNV. Inoculation of MV-susceptible mice with MVSchw-sEWNV induced both high levels of specific anti-WNV neutralizing antibodies and protection from a lethal challenge with WNV. Passive administration with antisera to MVSchw-sEWNV prevented WNV encephalitis in BALB/c mice challenged with a high dose of WNV. The present study is the first to report that a recombinant live attenuated vector based on an approved and widely used MV vaccine can protect against a heterologous, medically important pathogen.

Measles vaccine, a live attenuated strain of measles virus (MV), is one of the safest and most effective human vaccines available. Since the 1960s, it has been given to billions of children. Vaccination campaigns have been very effective in the control of outbreaks of MV in developed countries. However, because of inadequate distribution of the vaccine in developing countries, there are still 45 million cases of measles and 800,000 child deaths per year worldwide [1]. MV vaccine induces a life-long immunity after 1 or 2 low-dose inoculations [2, 3]. Persistence of antibodies and CD8 cells for as long as 25 years after vaccination has been documented [4]. MV vaccine is easily produced on a large scale in most countries and can be distributed at low cost. The MV genome is very stable, and reversion to pathogenicity has never been observed [3]. Moreover, MV replicates exclusively in the cytoplasm, ruling out the possibility of integration in host DNA. All these characteristics make live attenuated MV vaccine an attractive candidate vaccination vector that could provide safe and effective pediatric immunity against measles and, at the same time, against other infectious diseases. To this end, a reverse genetics system for MV has been established [5], allowing the production of recombinant MV with additional foreign genetic material. The MV vector has been shown to stably express a variety of genes or combinations of genes of large size over >12 passages [6–11]. This stability is likely due to the fact that there is little constraint on genome size for pleomorphic viruses with a helical nucleocapsid. Interestingly, because MV infects cells of the immune system—in particular, macrophages and dendritic cells—MV vectors deliver their foreign cargo directly to the most-efficient antigen-presenting cells.

We developed an MV vector based on the Schwarz strain, the safest and most widely used vaccine strain [12]. The vaccine rescued from the molecular clone has been shown to be as immunogenic as the parental vaccine in primates and mice susceptible to MV infection [12]. We recently produced recombinant MV expressing envelope (E) glycoproteins from HIV-1 that induced strong cellular immunity and neutralizing antibodies against MV and the HIV inserts [11]. Most importantly, preexisting immunity to the vector did not impair the immunogenic potential of the recombinant MV in mice and in macaques [11], leading to the possibility of using MV vector in an adult human population that has already been immunized.

In the present study, we evaluated whether immunization with an approved and widely used MV vaccine can protect against mosquito-borne West Nile virus (WNV). WNV is a member of the Flavivirus genus (Flaviviridae family) and caused the largest recognized epidemic of neuroinvasive human disease in North America [13, 14]. We generated a recombinant MV vector expressing the secreted form of the E (sE) glycoprotein of WNV. Adult mice are highly sensitive to low doses of WNV after peripheral inoculation [15, 16] and develop a neuroinvasive lethal disease similar to human disease [17]. Therefore, we evaluated the efficacy of recombinant MV in the mouse model.

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Materials and Methods

Cells and virus

Vero-NK (African green monkey kidney) cells were maintained in Dulbecco's modified Eagle medium (DMEM) Glutamax (Invitrogen) supplemented with 5% heat-inactivated fetal calf serum (FCS). Helper 293-3-46 cells (a gift from M. A. Billeter, Zurich University) used for viral rescue [5] were grown in DMEM/10% FCS and supplemented with 1.2 mg of G418/mL. The IS-98-ST1 strain of WNV (GenBank accession number AF481864) [16], a closely related variant of the NY99 strain [18], was propagated in mosquito (Aedes pseudoscutellaris) AP61 cell monolayers [16]. Purification on sucrose gradients and virus titration on AP61 cells by focus immunodetection assay (FIA) were performed as described elsewhere [16, 19].

Mouse antisera to WNV

Anti-WNV hyperimmune mouse ascitic fluid (HMAF) was obtained by repeated inoculation of adult mice with IS-98-ST1, followed by inoculation with sarcoma 180. Mouse polyclonal anti-WNV antibodies were obtained by inoculation of adult WNV-resistant BALB/c-MBT congenic mice with 103 focus-forming units (FFU) of IS-98-ST1, as described elsewhere [16]. Serum from WNV-immune mice was collected 1 month after priming.

Construction of pTM-MVSchw-sEWNV plasmid

The plasmid pTM-MVSchw, which contains an infectious MV cDNA corresponding to the anti-genome of the Schwarz MV vaccine strain, has been described elsewhere [12]. Genomic RNA of WNV was extracted from highly purified IS-98-ST1 virions and reverse transcribed by use of the Titan One-Step reverse-transcriptase polymerase chain reaction (RT-PCR) kit (Roche Molecular Biochemicals). An RT-PCR fragment encoding the internal E translocation signal (prM-151-prM-166), followed by the ectodomain and the stem region of the E protein (E-1-E-441), was generated by use of the forward primer 5′-TATCGTACGATGAGAGTTGTGTTTGTCGTGCTA-3′, which contains a BsiWI restriction site (underlined), and the reverse primer 5′-ATAGCGCGCTTAGACAGCCTTCCCAACTGA-3′, which contains a BssHII restriction site (underlined). A start and a stop codon were added at both ends. The sequence respects the “rule of six,” which stipulates that the number of nucleotides of the MV genome must be a multiple of 6 [20, 21]. After sequencing, the 1.4-kb cDNA containing the truncated E protein was inserted into BsiWI/BssHII-digested pTM-MVSchw-ATU2, which contains the additional transcription unit (ATU) between the phosphoprotein (P) and matrix (M) genes of the Schwarz MV genome [5, 12]. The resulting plasmid was designated as pTM-MVSchw-sEWNV.

Rescue of recombinant MVSchw-sEWNV from the cloned cDNA

Rescue of recombinant Schwarz MV from the plasmid pTM-MVSchw-sEWNV was performed by use of the helper-cell-based rescue system described by Radecke et al. [5] and modified by Parks et al. [22]. Briefly, 293-3-46 helper cells were transfected with 5 µg of pTM-MVSchw-sEWNV and 0.02 µg of pEMC-La expressing the MV polymerase L gene (a gift from M. A. Billeter, Zurich University). After overnight incubation at 37°C, a heat shock was applied for 2 h at 43°C, and transfected cells were transferred onto a Vero cell monolayer. Syncytia that appeared after 2–3 days of coculture were transferred to 35-mm wells of Vero cells and expanded in 75-cm2 and then 150-cm2 flasks, in DMEM/5% FCS. When syncytia reached 80%–90% confluence, the cells were scraped into a small volume of OptiMEM (Invitrogen) and frozen and thawed once. After low-speed centrifugation to pellet cellular debris, the supernatant, which contained virus, was stored at −80°C. The titer of MVSchw-sEWNV was determined by an endpoint limit-dilution assay on Vero cells. The TCID50 was calculated by use of the Kärber method.

Radioimmunoprecipitation assay (RIPA) and immunofluo rescence

Vero cells were starved for 1 h with DMEM without methionine and cysteine and labeled for 3 h with 250 µCi/mL Tran35S-label (both from ICN Biomedicals). Cells were lysed with RIPA buffer, and RIPA was performed as described elsewhere [23]. Samples were analyzed by 15% SDS-PAGE, by use of reducing conditions. Immunofluorescence staining was performed on infected Vero cells, as described elsewhere [24].

Mice experiments

Adult BALB/c mice were purchased from Janvier Laboratories. CD46+/− interferon (IFN)-α/β receptor−/− (CD46-IFNAR) mice were produced as described elsewhere [12]. Five-6-week-old CD46-IFNAR mice were inoculated intraperitoneally (ip) with either 104 or 106 TCID50 of recombinant MV. Pooled immune sera from immunized CD46-IFNAR mice were transferred ip into 6-week-old female BALB/c mice 1 day before challenge (serial dilutions of sera in 0.1 mL of Dulbecco's PBS/0.2% bovine serum albumin). Acute WNV challenge was performed by ip inoculation with neurovirulent IS-98-ST1 (ip LD50, 10), as described elsewhere [16]. The challenged mice were monitored daily for signs of morbidity and mortality. All experiments were approved by and conducted in accordance with the guidelines of the Office of Laboratory Animal Care at the Pasteur Institute.

Humoral response

Mice were bled via the periorbital route, and serum samples were heat inactivated for 30 min at 56°C. Anti-MV antibodies were detected by ELISA (Trinity Biotech). An anti-mouse antibody-horseradish peroxidase conjugate (Amersham) was used as the secondary antibody. The endpoint titer was calculated as the reciprocal of the last dilution eliciting twice the optical density (OD) of sera from nonimmunized mice. Anti-WNV antibodies were detected by ELISA, as described elsewhere [16], by use of microtitration plaques coated with 106 FFU of highly purified IS-98-ST1. Test sera were considered to be positive if the OD was twice the OD of sera from “empty” MVSchw-inoculated control mice. Peroxidase goat anti-mouse immunoglobulin (H+L) (Jackson ImmunoResearch) was used at a 1:4000 dilution, and peroxidase goat anti-mouse IgG (γ-chain specific) (Sigma) was used at a 1:20,000 dilution. Anti-WNV neutralizing antibodies were detected by use of a focus reduction neutralization test (FRNT). Serum samples from each mouse group were pooled and heat inactivated for 30 min at 56°C. Vero cells were seeded into 12-well plates (1.5 × 105 cells/well) for 24 h. Serum samples were serially diluted in DMEM Glutamax/2% FCS. Dilutions (0.1 mL) were incubated for 2 h at 37°C, under gentle agitation, with an equal volume of IS-98-ST1 containing ∼100 FFU of WNV IS-98-STI strain. Remaining infectivity was then assayed on Vero cell monolayers overlaid with DMEM Glutamax/2% FCS containing 0.8% (wt/vol) carboxy methyl cellulose. After 2 days of incubation at 37°C with 5% CO2, FIA was performed with anti-WNV HMAF, as described elsewhere [19]. The endpoint titer was calculated as the highest serum dilution tested that reduced the number of FFU by at least 90% (FRNT90).

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Results

Expression of the sE glycoprotein of WNV by recombinant MVSchw-sEWNV

The E glycoprotein has been shown to elicit protective immunity against WNV infection [25–31]. We previously observed that the growth of the MV vector expressing membrane-anchored viral glycoproteins was slightly delayed [11]. Therefore, we introduced the cDNA coding for the carboxyl terminal-truncated E glycoprotein lacking the transmembrane-anchoring region (residues E-1 through E-441; hereafter designated as sEWNV) of IS-98-ST1 [16] into the infectious cDNA of the Schwarz MV vaccine [12] (figure 1A). The WNV sequence was introduced into an ATU located between the P and the M genes in the MV genome. The recombinant MVSchw-sEWNV virus was produced after transfection of the corresponding plasmid into human helper cells, allowing the rescue of negative-stranded RNA paramyxoviruses [5], and then propagated in Vero cell cultures. Because MV vaccine is usually prepared as a crude clarified lysate from infected cells, the same basic process was used to produce MVSchw-sEWNV in the present study.

Figure 1
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Figure 1

Expression of the secreted form of the envelope (sE) glycoprotein of West Nile virus (WNV) by MVSchw-sEWNV recombinant measles virus (MV) in Vero cells. A, Schematic diagram of MVSchw-sEWNV and virus growth. The IS-98-ST1 cDNA coding for sEWNV was inserted into the Schwarz MV genome between the BsiW1 and BssHII sites of the additional transcription unit, at position 2. The MV genes are indicated: N (nucleoprotein), PVC (phosphoprotein and V and C proteins), M (matrix), F (fusion), H (hemagglutinin), and L (polymerase). δ, hepatitis delta virus ribozyme; hh, hammerhead ribozyme; T7, T7 RNA polymerase promoter; T7t, T7 RNA polymerase terminator. B, Growth curves of MV. Vero cells were infected with MVSchw (open box) or MVSchw-sEWNV (black box), at an MOI of 0.01 TCID50/cell. At various times after infection, titers of infectious virus particles were determined as described in Materials and Methods. C, Immunofluorescence staining of sE glycoprotein of WNV in syncytia of MVSchw-sEWNV-infected Vero cells fixed 36 h after infection. Cells were permeabilized with Triton X-100 (A and B) or not permeabilized (C and D) and then were immunostained by use of anti-WNV hyperimmune mouse ascitic fluid. Magnification, ×1000. No positive signal was observed in MVSchw-infected cells. D, Radioimmunoprecipitation assay showing the release of sEWNV from MVSchw-sEWNV-infected cells. Vero cells were either mock infected (MI) or infected with IS-98-ST1 at 5 focus-forming units/cell for 24 h (WNV), MVSchw at 0.1 TCID50/cell (MVSchw), or MVSchw-sEWNV at 0.1 TCID50/cell for 40 h (MVSchw-sEWNV). Radiolabeled supernatants and cell lysates were immunoprecipitated with specific anti-MV (α-MV) or anti-WNV (α-WNV) polyclonal antibodies. WNV envelope (E) glycoprotein (open arrowhead) and sEWNV (black arrowhead) are shown.

The growth of MVSchw-sEWNV in Vero cells was only slightly delayed, compared with that of empty Schwarz MV (MVSchw) (figure 1B). At 60 h after infection, the yield of MVSchw-sEWNV was comparable to that of MVSchw. The expression of sEWNV in MVSchw-sEWNV-infected Vero cells was determined by immunofluorescence and RIPA after 4 passages of MVSchw-sEWNV on Vero cells (figure 1C and 1D). At 40 h after infection, the cell surface of MVSchw-sEWNV-induced syncytia was clearly visualized by use of anti-WNV immune sera, indicating that sEWNV is transported along the compartments of the secretory pathway (figure 1C). RIPA analysis showed that anti-WNV antibodies recognized sEWNV (88% form of E) that migrated faster than did authentic E glycoprotein (figure 1D). A second protein, of smaller size, was also recognized. This form, which is also present at a lower level in lysates from WNV-infected cells, might correspond to posttranslational modification of the E glycoprotein. Interestingly, sEWNV was detected in the supernatants of MVSchw-sEWNV-infected Vero cells at 40 h after infection (figure 1D). Thus, MVSchw-sEWNV expresses a recombinant E glycoprotein that is secreted efficiently, even after 4 passages, confirming the stability of transgene expression in this system. Immunoblots confirmed that sEWNV accumulated in the culture medium of MVSchw-sEWNV-infected Vero cells (data not shown).

Induction of anti-WNV neutralizing antibodies

Genetically modified mice expressing the human CD46 MV receptor and lacking the IFN-α/β receptor [11, 32] (CD46-IFNAR) that are susceptible to MV [32] were used to assess the immune response induced by MVSchw-sEWNV. These mice develop cellular and humoral immune responses similar to those raised by competent mice [9, 11, 12, 33]. Groups of 6 CD46-IFNAR mice (5–6 weeks old) were inoculated ip with either 104 or 106 TCID50 of MVSchw-sEWNV. Each group received booster inoculations with the same dose 1 month later. As a control, CD46-IFNAR mice were inoculated with 106 TCID50 of empty MVSchw. One month after the first inoculation, specific anti-MV antibodies were detected in immune sera from all mice (table 1). Mice that received either dose of MVSchw-sEWNV displayed specific anti-WNV antibodies at a dilution of 1:40,000–1:60,000. One month after boosting, the titers of anti-WNV antibodies reached 1:500,000–1:900,000 (table 1) and were highly reactive with the E glycoprotein of WNV (figure 2). Isotype-specific ELISA showed that WNV-specific IgG antibodies were present in sera from MVSchw-sEWNV-inoculated mice 1 month after the first inoculation, and the titer was 10-fold higher 1 month after boosting (table 1). No anti-WNV antibodies were detected in sera from any control mice (table 1 and figure 2). These results show that 1 inoculation with MVSchw-sEWNV induces anti-WNV antibodies and that boosting 1 month after priming increases titers 12.5-18-fold.

Figure 2
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Figure 2

Recognition of the West Nile virus (WNV) envelope (E) glycoprotein by anti-MVSchw-sEWNV antibodies. Labeled cell lysates from WNV-infected Vero cells were immunoprecipitated with pooled immune sera (dilution, 1:100) from CD46+/− interferon-α/β receptor−/− mice inoculated with MVSchw (α-MV) or MVSchw-sEWNV (α-MVSchw-sEWNV), as described in the legend to figure 1D. Sera collected after the first or the second inoculation and after challenge (ac) were used. Anti-WNV immune sera (α-WNV) was used as a positive control. WNV structural glycoproteins prM and E and nonstructural proteins NS3, NS5, NS2A, and NS2B are shown.

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Table 1

Antibody response of CD46+/− interferon-α/β receptor−/− (CD46-IFNAR) mice to intraperitoneal inoculation with MVSchw-sEWNV.

Anti-WNV neutralizing activity was measured in immune sera from MVSchw-sEWNV-inoculated mice by use of an FRNT90 (table 1). The immune sera from WNV-resistant congenic mice [16] gave a FRNT90 titer of 50. The immune sera from CD46-IFNAR mice inoculated with empty MVSchw had no detectable neutralizing activity. Immunized CD46-IFNAR mice that received either 104 or 106 TCID50 of MVSchw-sEWNV elicited neutralizing antibodies with similar FRNT90 titers, and boosting increased titers from 10 to 200–300. These data show that mice inoculated twice with the recombinant live attenuated MV encoding the sE glycoprotein of IS-98-ST1 had high levels of anti-WNV antibody with neutralizing activity, regardless of the injected dose. The intracellular form of the E glycoprotein produced in MVSchw-sEWNV-infected cell culture (figure 1C) and contained in the crude vaccine preparation might have contributed to the immunogenicity. However, mice that received 104 TCID50 of MVSchw-sEWNV (the standard dose of MV vaccine used in humans) were injected with a 10−3 dilution of the infected crude cell lysate containing <1 µg of total proteins (determined by use of the Bradford assay). Such a low dose of recombinant MV has been proven to be effective. It is unlikely that intracellular sE antigen, which may represent as little as 0.1% of total proteins (data not shown), plays a major role in protection.

Protection of CD46-IFNAR mice against challenge with WNV

Mice that are completely unresponsive to IFN-α/β are highly susceptible to encephalitic flaviviruses [24, 34]. Indeed, we have previously shown that WNV infection of CD46-IFNAR mice was lethal within 3 days, instead of the 11 days in immunocompetent mice [24]. To assess whether the immunity induced by MVSchw-sEWNV could protect these compromised mice from WNV infection, groups of CD46-IFNAR mice that received 2 inoculations with either 104 or 106 TCID50 of MVSchw-sEWNV were inoculated ip with a lethal dose of WNV 1 month after boosting (table 2). Mice inoculated with empty MVSchw were used as controls. Remarkably, neither morbidity nor mortality was observed after challenge in mice inoculated with MVSchw-sEWNV, regardless of the dose used for immunization, whereas control mice died within 3–4 days. Mice that received 106 TCID50 of MVSchw-sEWNV were bled 3 weeks after challenge. The FRNT90 antibody response (titer, ∼100) was comparable to the prechallenge response. Notably, postchallenge immune sera did not react with WNV nonstructural proteins, such as NS3 and NS5, as shown by RIPA (figure 2), suggesting that no viral replication occurred after challenge with WNV. These data show that inoculation with MVSchw-sEWNV prevented WNV infection in highly susceptible animals.

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Table 2

Protection, by MVSchw-sEWNV, from a lethal challenge with West Nile virus in CD46+/− interferon-α/β receptor−/− (CD46-IFNAR) mice.

Protection of BALB/c mice after passive transfer of immune sera

Because antibody-mediated immunity is critical in protection against WNV infection [28, 30], we examined whether the passive transfer of sera from MVSchw-sEWNV-inoculated mice can protect adult BALB/c mice from WNV infection (table 3). Groups of 6 BALB/c mice (6 weeks old) received ip inoculations with various amounts of pooled immune sera collected from MVSchw-sEWNV-inoculated CD46-IFNAR mice 1 month after priming or boosting. One day later, the mice were challenged with 10 times the ip LD50 of IS-98-ST1 [16, 24]. BALB/c mice that received as little as 2 µL of sera from WNV-immune mice were protected from the challenge (table 3). In contrast, all mice that received 2 µL of serum from nonimmunized mice or from empty MVSchw-inoculated mice died within 11–12 days. Protective passive immunity was observed in all BALB/c mice after transfer of 2 µL of pooled sera from CD46-IFNAR mice inoculated once with 106 TCID50 of MVSchw-sEWNV. As little as 1 µL of this antisera induced a survival rate of 66%. Passive transfer of sera collected 1 month after 1 inoculation with 104 TCID50 induced a survival rate of 50%. Remarkably, the administration of 1 µL of immune sera from MVSchw-sEWNV-inoculated mice collected 1 month after boosting induced a survival rate of 100%. These results indicate that 1 inoculation with 106 TCID50 or 2 inoculations with 104 TCID50 of MVSchw-sEWNV elicited a protective humoral response. Because the amount of flavivirus inoculated during mosquito feeding is probably in the order of 102–104 infectious virus particles [35], we assessed the capacity of immune sera from MVSchw-sEWNV-inoculated mice to protect against a range of 102–105 FFU of IS-98-ST1. Groups of 6 BALB/c mice were passively inoculated with 2 µL of pooled immune sera from CD46-IFNAR mice inoculated twice with 104 TCID50 of MVSchw-sEWNV (table 3). Survival rates of 85%–100% were observed in mice that received immune sera from MVSchw-sEWNV-inoculated mice, regardless of the doses of IS-98-ST1 used for the challenges (ip LD50, 10–10,000). These data are consistent with the finding that the humoral response plays a critical role in protection against WNV infection and that neutralizing antibodies elicited by MVSchw-sEWNV effectively protect mice from lethal doses of WNV.

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Table 3

Protective ability of immune sera from MVSchw-sEWNV–inoculated mice.

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Discussion

Preventing WNV disease is a new public health priority in North America. Because there is no specific treatment for WNV disease, developing a vaccine is a priority [36–38]. Several immunization strategies have been evaluated in animal models. Inactivated WNV has been shown to generate a humoral response of low magnitude or poor quality, with a risk of immune enhancement of heterologous flavivirus infection [39]. Plasmid-encoding structural proteins of WNV elicited protective humoral and cellular immune responses [25, 40]. Chimeric WNV/yellow fever virus (YFV) and WNV/dengue virus type 4 (DV4) viruses bearing the NY99 WNV prM (the intracellular precursor of M) and E genes into the infectious clone backbone of YFV or DV4, respectively, have been constructed [41, 42]. Inoculation with these chimeric WNV-YF and WNV-DEN-4 viruses protected mice and prevented viremia in rhesus macaques challenged with WNV [43, 44]. Although the use of chimeric flaviviruses raises a legitimate safety concern [45], they are considered as candidates for evaluation in clinical trials [46].

In the present study, we have demonstrated, for the first time, that recombinant live attenuated MV is effective at preventing heterologous viral disease. Inoculation with a Schwarz MV vaccine-derived vector expressing the sE glycoprotein of WNV protected against WNV encephalitis in mice. The use of MVSchw-sEWNV as a WNV vaccine candidate offers major advantages over other immunization strategies. Unlike chimeric viral vectors, MVSchw-sEWNV is an authentic live attenuated MV expressing an additional viral gene. This greatly reduces the risk of changing the tropism and the pathogenicity of Schwarz MV vaccine, as well as the risk of recombination. Unexpectedly for an RNA virus, the MV genome is very stable, and MV vectors stably express foreign genes [6–11]. The presence of anti-MV immunity in nearly the entire adult human population would seem to restrict the use of MV recombinants to infants, a worthy goal in itself. However, revaccinating already-immunized individuals results in a boost of anti-MV responses, especially if aerosolized vaccine is used [47, 48], suggesting that the vaccine expresses its proteins in spite of preexisting immunity. Likewise, in infants given MV vaccine during the first year of life, the presence of maternal antimeasles antibodies has been shown to limit the induction of antimeasles antibodies but not of specific T cell responses [49, 50]. We reported that an MV-HIV recombinant virus induced anti-HIV antibodies in mice and macaques, even in the presence of preexisting anti-MV immunity [11]. This opens the possibility of using the live attenuated MV-derived vector to immunize adult humans.

Our data demonstrate that vaccination with a low dose of MVSchw-sEWNV induces high titers of WNV-specific IgG antibodies and that passive transfer of small amounts of antisera protects mice from WNV encephalitis. The antibody response is an essential component of protection against WNV encephalitis, and both IgM and IgG antibodies contribute to immunity [30, 51]. The long-term duration of anti-WNV-specific IgG antibodies is essential for protection against WNV [30, 52, 53]. Antibodies to MV are detected between 10 and 15 days after vaccination and last for many years [2]. In postvaccination long-lasting immunity (8–10 years), production of IgG antibody is maintained continuously at high levels [54]. Determining the longevity of protection induced by MVSchw-sEWNV is difficult in the mouse model. This concern is currently being investigated in the primate model. It was recently reported that CD8+ T cells may play an important role in eradicating WNV infection [55]. Studies are currently underway to determine the role of the cellular immune response in controlling WNV infection in MVSchw-sEWNV-inoculated animals.

Because of cross-species transmission, it is feared that WNV will become a recurrent zoonosis with repeated seasonal outbreaks in humans. MVSchw-sEWNV has the potential to elicit long-term immunity against both MV and WNV in children and adolescents, which might be naturally boosted in the case of an outbreak of WNV.

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Acknowledgment

We thank Martin Billeter for kindly providing the measles virus rescue system.

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Footnotes

  • Financial support: Pasteur Institute (grant PTR34); Centre National de la Scientifique (grant support); Agence Nationale de Recherches sur le SIDA (fellowship to C.L.).

  • Received April 8, 2004.
  • Accepted August 2, 2004.
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