Preliminary experiments demonstrated the ability of VSV- HA to elicit an antibody response to HA, including neutralizing antibodies to influenza A/WSN virus (and to VSVrwt [vector virus]), and to confer protection from a lethal influenza A/WSN virus challenge (data not shown). In these preliminary studies mice were inoculated via an intraperitoneal route with live or UV-inactivated VSV-HA or VSVrwt, given booster doses intraperitoneally 3 weeks after initial inoculation, and challenged intranasally with 50 μl of influenza A/WSN virus 2 weeks after boosting. Mice inoculated with live VSV-HA had neutralizing antibody titers of >1:2,000 to influenza A/WSN virus and were protected from lethal challenge. Mice receiving UV-inactivated VSV-HA (in the presence of adjuvant) were also protected from lethal challenge. Control mice receiving live or UV-inactivated VSVrwt did not produce neutralizing titers of antibody to influenza A/WSN virus (although they did produce neutralizing titers of antibody to VSV) and were not protected from influenza A/WSN virus challenge.
These preliminary data suggested that VSV-HA might be a useful tool in examining recombinant VSVs as potential recombinant vector vaccines. Although inoculation of mice through an intraperitoneal route and the use of adjuvant with the UV-inactivated virus are procedures often used in studying the efficacy of potential immunogens, it is not an ideal method for vaccine delivery. These drawbacks, along with the following three observations, contributed to the strategy employed in the present study: (i) VSV naturally infects at mucosal surfaces; (ii) influenza virus is naturally transmitted through the respiratory route; and (iii) a mouse model system exists for intranasal inoculations of mice with both viruses. We therefore examined the efficacy of VSV-HA in protecting mice from a lethal influenza A/WSN virus challenge when both inoculations and challenge were delivered intranasally.
Determination of a mouse 50% lethal dose (LD50) for the live recombinant vector VSVrwt.
When wild-type VSV (serotype Indiana) is administered to young mice (5 to 6 weeks old) via an intranasal route, the mice often develop hind-limb paralysis and die from a lethal encephalitis within 7 to 12 days after inoculation, with titers as low as 104
PFU/mouse proving fatal (our data and reference 9
). Therefore, before examining the efficacy of a recombinant VSV vaccine, we first addressed the pathogenicity of the vaccine vector.
To determine whether the recombinant VSVs derived from plasmid DNAs were pathogenic in mice, wild-type VSV Indiana (VSVI) or VSVrwt (recombinant VSV) were plaque purified. Four plaques of each virus were picked, viral stocks were grown, and titers were determined. Six-week-old female BALB/c mice were inoculated intranasally with VSVI or VSVrwt in a total volume of 25 μl. Mice were weighed and observed daily for signs of pathogenesis as indicated by weight loss, paralysis, or death. A graphic representation of the average daily weights of mice inoculated with VSVI, VSVrwt, or medium only is presented in Fig. , in which numbers above the x axis indicate the number of mice dying that day.
FIG. 1 Effects of viral inoculation on average weight and survival. (A) Twelve 6-week-old female BALB/c mice were divided into four groups of three mice each. Four plaque-purified viral stocks of VSVI were administered intranasally (one plaque-purified stock/group (more ...)
Mice inoculated with VSVI experienced much greater pathogenesis than did mice inoculated with VSVrwt as indicated by initial weight loss, time to weight loss recovery (if any), onset of hind-limb paralysis, and lethality. Of the mice receiving VSVI, 75% suffered weakness and paralysis within 4 to 7 days and subsequently died within 10 days. The 25% of VSVI-inoculated mice that survived all received virus from the same plaque-purified stock, perhaps indicating a slight decrease in pathogenesis for that particular stock. Two of these three surviving mice showed signs of increased pathogenesis compared to VSVrwt-inoculated mice. These two mice lost 20.7 and 35.4% of initial body weight and began regaining weight at 9 and 10 days postinoculation, respectively. The other mouse lost 15.4% of its initial body weight and began regaining weight at 5 days postinoculation (similar to mice receiving VSVrwt). No paralysis or death was observed in mice receiving VSVrwt. The average percent loss of initial body weight in mice receiving VSVrwt was 20.7% ± 3.1%, and most mice began regaining initial weight loss within 4 to 6 days of inoculation. As a control for average daily weights, four mice were inoculated with DMEM only.
From our own data and from lethality data of VSVI
reported by Forger et al. (9
), a mouse LD50
of approximately 106
PFU/mouse was determined for intranasal inoculations of BALB/c mice (5 to 6 weeks old). The lethality of VSVI
varies slightly, depending on the virus preparation and the total volume of the virus inoculum. No LD50
has been reached for mice inoculated with VSVrwt under the same conditions even at titers that are 10-fold higher (107
PFU/mouse) than the LD50
. The attenuation of VSVrwt is apparent although further attenuation (one in which mice lack weight loss associated with pathogenesis) would be ideal. The attenuation of VSVrwt compared to VSVI
, however, indicates that VSVrwt-based vectors may potentially be used as a recombinant vaccine vectors.
Immunogenicity and vaccine efficacy of recombinant VSV-HA.
Having determined that intranasal inoculation with VSVrwt vector at titers of ≤107 PFU/mouse was not lethal, we examined the immunogenicity of recombinant VSV-HA. VSV-HA was administered at three titers (104, 105, and 106 PFU/mouse). Each titer was examined for its ability to raise neutralizing antibodies to influenza A/WSN virus and was further evaluated for its efficacy in protecting mice from lethal influenza A/WSN virus challenge. Mice were similarly inoculated with VSVrwt at matching titers or with DMEM as control groups.
Six-week-old female BALB/c mice were lightly anesthetized with Metofane and inoculated intranasally with live VSV-HA or VSVrwt in a total volume of 25 μl. Mice were observed daily for signs of paralysis and/or death, and none occurred. However, mice did appear somewhat less active and less well groomed during the first few days immediately following the initial inoculations. At day 18 postinoculation two mice per group were bled. Sera from these bleeds were pooled for each group and heat inactivated. Mice receiving VSV-HA virus produced antibodies to both VSV and influenza A/WSN virus as determined by indirect immunofluorescence assays (IFA) to VSV-infected BHK cells and to influenza A/WSN virus-infected MDBK cells, respectively. Mice inoculated with VSVrwt produced antibodies to VSV only. Sera were also assayed for their ability to neutralize influenza A/WSN virus by using a 50% plaque reduction assays on MDBK cells (Table ). Sera from mice inoculated with VSV-HA contained neutralizing antibodies to influenza A/WSN virus at titers of 1:512, indicating that intranasal inoculation with recombinant VSV-HA raised a systemic immune response to the heterologous influenza A/WSN virus when administered through a mucosal route. Sera collected from mice prior to inoculation showed no antibodies to either VSV or influenza A/WSN by IFA or by neutralization assays.
TABLE 1 Antibody response and percentsurvival
Mice were given booster doses 21 days after initial inoculation and observed for an additional 14 days. As before, paralysis and/or death were not observed in any mice, and all mice appeared healthy. At day 32, mice were bled and sera were tested for antibodies by IFA and neutralization assays. The results (Table ) were similar to those obtained at 18 days. At day 35, mice were challenged intranasally with a lethal dose of influenza A/WSN virus (10 LD100 in 50 μl). Mice were observed for an additional 14 days after influenza A/WSN virus challenge. Within 5 days of influenza A/WSN virus challenge, all mice inoculated with VSVrwt had died. No mice inoculated with VSV-HA died or showed any signs of sickness. These data show that recombinant VSVs delivered intranasally are not only efficacious in raising systemic immunity but also completely protect mice from morbidity and death associated with a lethal challenge when the recombinant VSV expresses a foreign antigen corresponding to the challenge virus. These data also show that VSV-HA titers as low as 104 PFU/mouse are sufficient to protect mice from a lethal influenza A/WSN virus challenge.
Pathogenicity, immunogenicity, and vaccine efficacy of recombinant VSV-HA.
Several observations led to further experiments examining the potential of recombinant VSVs as vaccine vectors. Taken together, the ability of low titers of VSV-HA to protect from lethal influenza A/WSN virus challenge and the reduced pathogenicity of the VSV vector compared to VSVI suggested that we should examine the pathogenicity of the recombinant VSV-HA and VSVrwt at lower titers. Therefore, pathogenicity (as indicated by weight loss), immunogenicity (as indicated by antibody production), and vaccine efficacy (as indicated by an ability to protect mice from lethal influenza A/WSN virus challenge) were examined in mice inoculated with VSV-HA or VSVrwt at titers of 5 × 104 PFU/mouse.
Mice were inoculated intranasally with VSV-HA, VSVrwt, or DMEM only on day 0 and weighed daily. At 18 days after initial inoculation, two mice per group were bled and sera were pooled and heat inactivated. These pooled sera were assayed for neutralizing titers to influenza A/WSN virus by 50% plaque reduction assays on MDBK cells. Mice inoculated with VSV-HA had neutralizing serum titers of 1:640 of antibody to influenza A/WSN virus (Table ). Mice were boosted with recombinant VSVs (or DMEM) on day 21, and sera were subsequently collected, pooled, and assayed for neutralizing titers of antibody to influenza A/WSN virus on day 32. Mice inoculated with VSV-HA had neutralizing serum titers of 1:2,560 of antibody to influenza A/WSN virus. No neutralizing antibodies to influenza A/WSN virus were detected in mice prior to inoculation or in mice inoculated with VSVrwt or DMEM (Table ). Mice inoculated with recombinant VSVs were challenged with a lethal dose of influenza A/WSN virus (50 μl) on day 35. Mice inoculated and boosted with DMEM were boosted with 50 μl of DMEM on day 35 and maintained as a weight control group.
TABLE 2 Neutralization titers and percent survival of mice challenged with influenza A/WSNvirus
Mice receiving initial inoculations of recombinant VSVs showed signs of pathogenesis as indicated by weight loss. This is demonstrated in Fig. A and B by the dips in daily weight averages (days 0 to 5). This initial weight loss was similar in both VSVrwt- and VSV-HA-inoculated mice. The percentages of initial body weight loss were 19.3 ± 4.5 and 18.1 ± 3.4 in mice inoculated with VSVrwt and VSV-HA, respectively. In contrast, the percentage of initial body weight loss in DMEM-inoculated mice was only 4.4 ± 2.0. After the boosting treatment, no significant weight loss occurred in any mice (days 21 to 23), indicating immunity to the vector. On day 35 mice were challenged with a lethal dose of influenza A/WSN virus. VSV-HA-inoculated mice were completely protected from lethal challenge as indicated by the absence of significant weight loss (Fig. B) and 100% survival (Table ). In contrast, control mice inoculated with VSVrwt were not protected from challenge with influenza A/WSN virus. Two of these five control mice died within 5 days of influenza A/WSN virus challenge (Table ). Although three mice did survive the challenge in this particular experiment, these mice were extremely ill, as indicated by extensive weight loss (26.1 ± 9.4%), as well as by ruffled coats and inactivity, and did not begin recovering weight until day 44 (9 days after challenge).
FIG. 2 Effects of vaccination and challenge on average weight and survival. (A) Mice were inoculated intranasally with VSVrwt at 5 × 104 PFU/mouse on day 0. Mice were boosted with an equal amount of inoculum on day 21. Mice were challenged with a lethal (more ...)
In an experiment parallel to those described above, we examined the pathogenicity, immunogenicity, and efficacy of VSV-HA in mice receiving a single intranasal inoculation on day 0 and challenged with a lethal dose of influenza A/WSN virus on day 21. These mice showed an initial weight loss of 17.6 ± 2.3% from the vector inoculation (Fig. C). Sera were collected, pooled, and assayed for neutralizing titers of antibody to influenza A/WSN virus at day 18 (Table ). Mice were then challenged with a lethal dose of influenza A/WSN virus on day 21. These results showed that, even after a single inoculation, VSV-HA-immunized mice were completely protected from lethal influenza A/WSN virus challenge. Complete protection was indicated by negligible weight loss (Fig. C, days 21 to 23), the presence of neutralizing titers of antibody to influenza A/WSN virus, and 100% survival (Table ).
VSV-HA infection protects against influenza virus-induced bronchopneumonia.
To further assess the immune reaction against VSVrwt and VSV-HA, we inoculated two mice each as described above with 5 × 104 PFU/mouse of VSVrwt, VSV-HA, or DMEM alone and examined the spleens of the mice 7 days after initial infection for reactive germinal centers. The spleens from mice infected with VSVrwt and VSV-HA showed active germinal centers, indicating an active immune response, whereas spleens from mice inoculated with DMEM showed no active germinal centers. Lung sections taken from the same mice showed lymphocytic infiltrates in the bronchi of VSVrwt and VSV-HA recipients, indicating possible viral infection; lung sections from DMEM-inoculated mice showed no infiltrates. No signs of pneumonia (i.e., the alveolar spaces were clear) were found in any lung sections (data not shown).
The survival analysis presented above indicated that inoculation of mice with VSV-HA led to complete resistance against influenza A/WSN virus challenge, including protection from influenza A/WSN virus-induced pneumonia. To assess this directly, we examined the lungs of VSV-HA-inoculated mice challenged with influenza A/WSN virus. Two mice each were inoculated with PBS, VSV-HA, or VSVrwt on day 0, boosted with the same agent on day 21, and then challenged with influenza A/WSN virus on day 35. At 3 days after challenge, mice were sacrificed and the lungs were examined for histopathology. Lung sections from mice inoculated with VSVrwt (Fig. D) or PBS (Fig. B) and challenged with influenza A/WSN virus showed evidence of acute viral bronchopneumonia, as manifested by cytopathic changes in the bronchial epithelium and cellular debris in the bronchial lumens and alveolar spaces. Additional signs of infection and reaction included a marked peribronchial lymphocytic infiltrate and, in the pulmonary vessels, a marked thickening of both the vessel wall and its endothelial lining (Fig. B and D). In contrast, lung sections of mice inoculated with VSV-HA and challenged with influenza A/WSN virus showed an intact bronchial epithelium, no thickening of the vessels, and clear alveolar spaces (Fig. C). Lungs from mice inoculated with DMEM, which were boosted and challenged with DMEM alone, also showed no signs of pathology (Fig. A).
FIG. 3 Histopathology in lungs in mice 3 days after WSN virus challenge. Mice were inoculated and boosted with DMEM (A), PBS (B), VSV-HA (C), or VSVrwt (D) and then challenged with either influenza A/WSN virus (B, C, and D) or DMEM (A). Abbreviations: BE, bronchial (more ...) Pathogenicity and immunogenicity of recombinant VSV-CT1 and VSV-CT9.
These experiments demonstrated the immunogenicity and complete efficacy of a recombinant VSV vaccine expressing influenza A/WSN/33 HA protein in protecting mice from a lethal influenza A/WSN virus challenge. However, the pathogenicity associated with the immunizing vector remained a concern. Although this initial pathogenicity is limited (mice began regaining initial weight loss within 6 days of immunization), further attenuation of the VSV vaccine vector was addressed. To this end, we examined recombinant VSVs which have previously indicated attenuation in vitro (reduced plaque size and reduced viral titers) compared to VSVrwt. Specifically, two recombinant VSV constructs, VSV-CT1 and VSV-CT9 (21
), which have deletions truncating the glycoprotein cytoplasmic tails from 29 amino acids to 1 and 9 amino acids, respectively, were examined for pathogenicity and immunogenicity in the mouse model system.
Six-week-old female BALB/c mice were lightly anesthetized with Metofane and inoculated intranasally with VSVrwt, VSV-CT1, VSV-CT9, or DMEM in a total volume of 25 μl. Mice were observed and weighed daily (Fig. ). At 14 days after inoculation two mice per group were bled, and sera were pooled within each group. Again, at 28 days after inoculation two mice per group were bled, and sera were pooled within each group. These pooled sera were heat inactivated and assayed for neutralizing titers of antibody to VSVrwt by complete inhibition of the CPE in BHK cells (Table ). Neutralization titers are reported as ranges obtained in multiple assays.
FIG. 4 Effects of inoculation with attenuated viral vectors on average daily weights. (A) Six-week-old female BALB/c mice were inoculated intranasally with VSV-CT1 at 105 PFU/mouse in a 25-μl total volume. Control mice (DMEM-0) received 25 μl (more ...)
TABLE 3 Neutralizing antibody titers toVSVrwt
Mice inoculated with VSVrwt (105 PFU/mouse) showed signs of initial pathogenesis as indicated by weight loss as previously observed. However, in stark contrast, mice inoculated with VSV-CT1 (105 PFU/mouse) experienced negligible weight loss, a finding comparable to that observed in DMEM-inoculated control mice (Fig. A). Furthermore, mice inoculated with VSV-CT1 or DMEM remained active and well groomed in the days following inoculation. Mice receiving VSVrwt had reduced activities and were poorly groomed on days 1 to 5, corresponding to the days of weight loss (Fig. C).
Mice inoculated with VSV-CT9 (105 PFU/mouse) showed delayed and reduced pathogenesis compared to VSVrwt-inoculated mice. The amount of weight loss and the rate of weight loss in VSV-CT9-inoculated mice were not as great as those seen in VSVrwt-inoculated mice but were still measurably significant compared to the DMEM-inoculated mice (Fig. B). Although pathogenesis was eliminated for VSV-CT1-inoculated mice and reduced for VSV-CT9-inoculated mice, sera collected from both groups at 14 and 28 days after inoculation contained neutralizing antibodies to VSVrwt. These neutralizing antibody titers were similar to those obtained in VSVrwt-inoculated mice (Table ). Sera from DMEM-inoculated mice and sera collected from mice 3 days prior to inoculation did not contain neutralizing titers of antibodies to VSVrwt at dilutions of 1:8 (the lowest dilution assayed).