Attenuated Salmonella enterica serovar Typhi and Shigella flexneri 2a strains mucosally deliver DNA vaccines encoding measles virus hemagglutinin, inducing specific immune responses and protection in cotton rats

Abstract

Measles remains a leading cause of child mortality in developing countries. Residual maternal measles antibodies and immunologic immaturity dampen immunogenicity of the current vaccine in young infants. Because cotton rat respiratory tract is susceptible to measles virus (MV) replication after intranasal (i.n.) challenge, this model can be used to assess the efficacy of MV vaccines. Pursuing a new measles vaccine strategy that might be effective in young infants, we used attenuated Salmonella enterica serovar Typhi CVD 908-htrA and Shigella flexneri 2a CVD 1208 vaccines to deliver mucosally to cotton rats eukaryotic expression plasmid pGA3-mH and Sindbis virus-based DNA replicon pMSIN-H encoding MV hemagglutinin (H). The initial i.n. dose-response with bacterial vectors alone identified a well-tolerated dosage (1 x 10(9) to 7 x 10(9) CFU) and a volume (20 micro l) that elicited strong antivector immune responses. Animals immunized i.n. on days 0, 28, and 76 with bacterial vectors carrying DNA plasmids encoding MV H or immunized parenterally with these naked DNA vaccine plasmids developed MV plaque reduction neutralizing antibodies and proliferative responses against MV antigens. In a subsequent experiment of identical design, cotton rats were challenged with wild-type MV 1 month after the third dose of vaccine or placebo. MV titers were significantly reduced in lung tissue of animals immunized with MV DNA vaccines delivered either via bacterial live vectors or parenterally. Since attenuated serovar Typhi and S. flexneri can deliver measles DNA vaccines mucosally in cotton rats, inducing measles immune responses (including neutralizing antibodies) and protection, boosting strategies can now be evaluated in animals primed with MV DNA vaccines.

FIG. 1.

Schematic representation of eukaryotic expression vectors pGA3-mH and pMSIN-H encoding MV H. (A) The pGA3-mH vector contains a CMV immediate-early promoter plus intron A for initiating transcription of eukaryotic inserts and bovine growth hormone polyadelylation signal (BGH polyA) for termination of transcription. The vector also contains the ColE1 origin of replication for prokaryotic replication, as well as the kanamycin resistance gene (Kan^r) for selection in antibiotic media. The T0 terminator has been placed 3′ to Kan^r to increase the stability of eukaryotic inserts. The MV H insert was cloned into pGA3 vector by using HindIII and BamHI restriction endonuclease sites. (B) Plasmid pMSIN-H is a Sindbis virus-based vector with the MV H gene cloned as an XhoI-NotI fragment. The replicon contains the ColE1 origin of replication and Kan^r marker. Replication of alphavirus genome is directed by the CMV promoter, whereas the expression of MV H is under the control of the subgenomic promoter.

FIG. 2.

IgG anti-LPS responses elicited in serum in cotton rats immunized i.n. with various doses of vaccine strain serovar Typhi (upper panel) or S. flexneri 2a (lower panel). Cotton rats were inoculated i.n. with 7.4 × 10⁷ to 7.4 × 10⁹ CFU of CVD 908-htrA or 3.0 × 10⁷ to 3.0 × 10⁹ CFU of CVD 1208 suspended in a 20-μl volume. A control group received 20 μl of PBS i.n. IgG anti-LPS titers were measured by ELISA in serum samples collected 21 days after immunization. Bars indicate the GM titers ± the SE from four animals. ✽, Significant increases (P < 0.05) in antibody titers compared to preimmunization levels and control group. EU, ELISA unit(s).

FIG. 3.

IgG responses in serum to serovar Typhi LPS and TT fragment C in cotton rats immunized i.n. with CVD 908-htrA(pTETlpp) and CVD 1208(pTETlpp). Cotton rats received one dose of 2.2 × 10⁸ to 2.2 × 10⁹ CFU of CVD 908-htrA(pTETlpp) or 7.4 × 10⁷ to 7.4 × 10⁸ of CVD 1208(pTETlpp). Titers of antibody to serovar Typhi, S. flexneri 2a LPS, and TT fragment C were measured by ELISA in serum samples collected 21 days after immunization. GM titers ± the SE from four animals are shown. ✽, Significant increases (P < 0.05) in antibody titers after immunization compared to preimmunization levels and control group. EU, ELISA unit(s).

FIG. 4.

IgG responses in serum to serovar Typhi LPS and TT fragment C in cotton rats immunized i.n. with CVD 908-htrA(pTETlpp). Cotton rats were immunized i.n. on days 0 and 28 (indicated by arrows) with 2 to 5 × 10⁹ CFU of CVD 908-htrA or CVD 908-htrA(pTETlpp). Curves indicate GM titers ± the SE from four animals. Titers were measured by ELISA in serum samples collected on days 0, 14, 28, 42, and 56 after primary immunization. ✽, Significant increases (P < 0.03) in antibody titers after immunization compared to preimmunization levels. EU, ELISA unit(s).

FIG. 5.

IgG responses in serum to MV H antigen in cotton rats and mice immunized with MV H DNA vaccine plasmids. Cotton rats were immunized i.m. on days 0, 28, and 56 with 100 μg of pGA3-mH or pMSIN-H in a volume of 100 μl. IgG responses to MV H antigen in serum were measured by ELISA and PRN assay on days 0, 28, 56, and 84. Mice were immunized in a similar way but received only two doses (on days 0 and 28). IgG responses to MV H antigen in serum were measured by ELISA and PRN assay on days 0, 28, and 56. Negative control groups included animals immunized with 100 μg of empty plasmids (pGA3 and pSINCP) or PBS. GM titers ± the SE from four to five animals are indicated. The numbers of animals that seroconverted within each group (i.e., that manifested a fourfold increase in antibody titers compared to preimmunization levels) are indicated. ✽, Significant increases (P < 0.05) in antibody titers after immunization. EU, ELISA unit(s).

FIG. 6.

Antibody responses to MV as measured by PRN assay. Groups of cotton rats were immunized i.n. on days 0, 28, and 76 with 4 × 10⁸ to 5 × 10⁹ CFU/20 μl of CVD 908-htrA(pGA3-mH), CVD 908-htrA(pMSIN-H), CVD 1208(pGA3-mH), and CVD 1208(pMSIN-H). Cotton rat groups serving as negative controls received CVD 908-htrA or CVD 1208 without plasmids and PBS i.n. Positive control groups included animals immunized i.m. with 100 μl (1 μg/μl) of plasmids pGA3-mH or pMSIN-H. PRN antibody titers were measured on days 0 (□), 28 (), 65 (), and 104 (▪). Responses are expressed as GM titers ± the SE from four to five animals in each group. The numbers of animals that seroconverted, i.e., manifested a fourfold increase in antibody titers, after the third dose within each group are indicated. ✽, Significant increases (P < 0.05) in antibody titers after immunization compared to preimmunization levels and control groups.

FIG. 7.

Proliferative responses against MV H antigen. Splenocytes from cotton rats immunized i.n. with three doses of CVD 908-htrA and CVD 1208 alone or carrying MV DNA vaccine plasmids, and i.m. with plasmids pGA3-mH or pMSIN-H, as described in Fig. 6, were incubated in vitro in the presence of MV H antigen or bovine serum albumin (BSA) as control. Responses were studied 4 months after primary immunization. Bars indicate peak responses within the range of concentrations tested (0.01 to 10 μg/ml for BSA and 1/20 to 1/1/80,000 for MV H antigen) and are expressed as the stimulation index ± the SE of triplicate wells. ✽, Significant responses (P < 0.05) compared to control groups. The results shown are representative of two separate experiments.

FIG. 8.

i.n. challenge of immunized cotton rats with wild-type MV. Groups of four to five cotton rats were immunized i.n. with 2 to 7 × 10⁹ CFU of CVD 908-htrA and CVD 1208 alone or carrying plasmids pGA3-mH and pMSIN-H; i.m. with 100 μl (1 μg/μl) of plasmids pGA3-mH and pMSIN-H and i.n. with PBS (control) as described in Fig. 6. One month after the third dose, animals were challenged i.n. with wild-type MV (10⁷ PFU in 100 μl). Pulmonary virus was measured in lung homogenates 4 days after challenge. The results are reported as the mean log₁₀ TCID₅₀/g of lung tissue ± the SE. The data represent the average for four to five animals. Significant differences between groups, when present, are indicated.