Protective immunity against a lethal respiratory Yersinia pestis challenge induced by V antigen or the F1 capsular antigen incorporated into adenovirus capsid

Abstract

The aerosol form of the bacterium Yersinia pestis causes pneumonic plague, a rapidly fatal disease that is a biothreat if deliberately released. At present, no plague vaccines are available for use in the United States, but subunit vaccines based on the Y. pestis V antigen and F1 capsular protein show promise when administered with adjuvants. In the context that adenovirus (Ad) gene transfer vectors have a strong adjuvant potential related to the ability to directly infect dendritic cells, we hypothesized that modification of the Ad5 capsid to display either the Y. pestis V antigen or the F1 capsular antigen on the virion surface would elicit high V antigen- or F1-specific antibody titers, permit boosting with the same Ad serotype, and provide better protection against a lethal Y. pestis challenge than immunization with equivalent amounts of V or F1 recombinant protein plus conventional adjuvant. We constructed AdYFP-pIX/V and AdLacZ-pIX/F1, E1(-), E3(-) serotype 5 Ad gene transfer vectors containing a fusion of the sequence for either the Y. pestis V antigen or the F1 capsular antigen to the carboxy-terminal sequence of pIX, a capsid protein that can accommodate the entire V antigen (37 kDa) or F1 protein (15 kDa) without disturbing Ad function. Immunization with AdYFP-pIX/V followed by a single repeat administration of the same vector at the same dose resulted in significantly better protection of immunized animals compared with immunization with a molar equivalent amount of purified recombinant V antigen plus Alhydrogel adjuvant. Similarly, immunization with AdLacZ-pIX/F1 in a prime-boost regimen resulted in significantly enhanced protection of immunized animals compared with immunization with a molar-equivalent amount of purified recombinant F1 protein plus adjuvant. These observations demonstrate that Ad vaccine vectors containing pathogen-specific antigens fused to the pIX capsid protein have strong adjuvant properties and stimulate more robust protective immune responses than equivalent recombinant protein-based subunit vaccines administered with conventional adjuvant, suggesting that F1-and/or V-modified capsid Ad-based recombinant vaccines should be considered for development as anti-plague vaccines.

FIG. 1.

Capsid-modified Ad vectors. The constructs used in this study are replication-defective E1^–,E3^– serotype 5 Ad gene transfer vectors containing (5′ to 3′) the cytomegalovirus immediate early promoter/enhancer (CMV), a marker gene, and the simian virus 40 poly(A) stop signal. Top: Ad5 genome. Middle: AdYFP-pIX/V expresses YFP as a transgene and has the V antigen coding sequence fused in frame to the C terminus of protein IX. Bottom: AdLacZ-pIX/F1 expresses LacZ as a transgene and has the coding sequence for the F1 capsular antigen fused in frame to the C terminus of protein IX. Expression of the pIX/V or pIX/F fusion protein is under the control of the pIX promoter.

FIG. 2.

Detection of V antigen or F1 antigen on the surface of capsid-modified Ad vectors by Western analysis. (A) Purified AdYFP-pIX/V virions, purified AdYFP virions as a negative control, or a molar equivalent amount of purified recombinant V antigen that corresponds to the amount of V antigen displayed on the capsid of AdYFP-pIX/V were assessed by Western analysis with an anti-V antigen polyclonal antiserum. Lane 1, AdYFP, 10¹⁰ pu; lane 2, AdYFP-pIX/V, 10¹⁰ pu; lane 3, purified recombinant V antigen, 0.15 μg. (B) Purified AdLacZ-pIX/F1 virions, purified AdLacZ virions as a negative control, or a molar equivalent amount of purified recombinant V antigen that corresponds to the amount of V antigen displayed on the capsid of AdYFP-pIX/V were assessed by Western analysis with a polyclonal rabbit F1 antibody. Lane 4, AdLacZ, 10¹⁰ pu; lane 5, AdLacZ-pIX/F1, 10¹⁰ pu; lane 6, purified recombinant F1 antigen, 0.05 μg. The molecular weights of pIX/V, pIX/F1, V antigen, and F1 are indicated.

FIG. 3.

Anti-V antigen antibodies and survival of mice immunized in a prime–boost regimen with AdYFP-pIX/V or V antigen/Alhydrogel. BALB/c mice (n = 8 to 16 per group) were immunized with AdYFP-pIX/V or purified V antigen/Alhydrogel. A subset of animals received a single repeat administration of the same immunogens at the same dose 4 weeks after primary immunization. (A) Serum anti-V antigen titers, AdYFP-pIX/V (10⁹ particle units, pu). Six weeks after the prime immunization serum antibody levels were measured in an anti-V antigen-specific ELISA (p < 0.0001, AdYFP-pIX/V unboosted or boosted vs. AdYFP). (B) Serum anti-V antigen titers, purified recombinant V antigen (15 ng, the molar equivalent amount of protein present in 10⁹ pu of AdYFP-pIX/V) with Alhydrogel (p < 0.009, V antigen unboosted or boosted vs. naive). (C) Survival, AdYFP-pIX/V. Six weeks after prime immunization with 10⁹ pu of AdYFP-pIX/V, mice were challenged with a lethal dose of Yersinia pestis CO92, and survival of the animals was monitored for 14 days. Naive mice and mice that received AdYFP (10¹¹ pu) intramuscularly were included as negative controls (p = 0.1437, AdYFP-pIX/V vs. AdYFP; p < 0.0001, AdYFP-pIX/V plus boost versus AdYFP). (D) Survival, V antigen/Alhydrogel. Naive mice were used as negative controls (p = 0.0093, V antigen vs. naive; p = 0.0248, V antigen plus boost vs. naive).

FIG. 4.

Anti-V antigen antibodies and survival of mice immunized in a prime–boost regimen with AdYFP-pIX/V or V antigen/Alhydrogel. BALB/c mice (n = 16 to 24 per group) were immunized with AdYFP-pIX/V or purified V antigen/Alhydrogel. A subset of animals received a single repeat administration of the same immunogens at the same dose 4 weeks after primary immunization. (A) Serum anti-V titers, AdYFP-pIX/V (10¹¹ particle units, pu). Six weeks after the prime immunization serum antibody levels were measured in an anti-V antigen-specific ELISA (p < 0.00007, AdYFP-pIX/V unboosted or boosted vs. AdYFP). (B) Serum anti-V titers purified recombinant V antigen (1.5 μg, the molar equivalent amount of protein present in 10¹¹ pu of AdYFP-pIX/V) with Alhydrogel (p < 0.0005, V antigen unboosted or boosted vs. naive). (C) Survival, AdYFP-pIX/V. Six weeks after prime immunization with 10¹¹ pu of AdYFP-pIX/V, mice were challenged with a lethal dose of Yersinia pestis CO92, and survival of the animals was monitored for 14 days. Naive mice and mice that received AdYFP (10¹¹ pu) intramuscularly were included as negative controls (p < 0.0001, AdYFP-pIX/V vs. AdYFP; p < 0.0001, AdYFP-pIX/V plus boost vs. AdYFP). (D) Survival, V antigen/Alhydrogel. Naive mice were used as negative controls (p = 0.3014, V antigen vs. naive; p < 0.0001, V antigen plus boost vs. naive).

FIG. 5.

Anti-F1 antibodies and survival of mice immunized in a prime–boost regimen with AdLacZ-pIX/F1 or F1/Alhydrogel. BALB/c mice (n = 11 to 22 per group) were immunized with AdLacZ-pIX/F1 or purified F1/Alhydrogel. A subset of animals received a single repeat administration of the same immunogens at the same dose 4 weeks after primary immunization. (A) Serum anti-F1 titers, AdLacZ-pIX/F1 (10⁹ particle units, pu). Six weeks after the prime immunization serum antibody levels were measured in an anti-F1-specific ELISA (p < 0.01, AdLacZ-pIX/F1 unboosted or boosted vs. AdLacZ). (B) Serum anti-F1 titers, purified recombinant F1 (5.1 ng, the molar equivalent amount of protein present in 10⁹ pu of AdLacZ-pIX/F1) with Alhydrogel (p < 0.01, F1 unboosted or boosted vs. naive). (C) Survival, AdLacZ-pIX/F1. Six weeks after prime immunization with 10⁹ pu of AdLacZ-pIX/F1, mice were challenged with a lethal dose of Yersinia pestis CO92, and survival of the animals was monitored for 14 days. Naive mice and mice that received AdLacZ (10¹¹ pu) intramuscularly were included as negative controls (p < 0.0001, AdLacZ-pIX/F1 vs. AdLacZ; p < 0.0001, AdLacZ-pIX/F1 plus boost vs. AdLacZ). (D) Survival, F1/Alhydrogel. Naive mice were used as negative controls (p < 0.0007, F1 vs. naive; p = 0.0044, F1 plus boost vs. naive).

FIG. 6.

Anti-F1 antibodies and survival of mice immunized in a prime–boost regimen with AdLacZ-pIX/F1 or F1/Alhydrogel. BALB/c mice (n = 15 to 31 per group) were immunized with AdLacZ-pIX/F1 or purified F1/Alhydrogel. A subset of animals received a single repeat administration of the same immunogens at the same dose 4 weeks after primary immunization. (A) Serum anti-F1 titers, AdLacZ-pIX/F1 (10¹¹ particle units, pu). Six weeks after the prime immunization serum antibody levels were measured by anti-F1-specific ELISA (p < 0.0003, AdLacZ-pIX/F1 unboosted or boosted vs. AdLacZ). (B) Serum anti-F1 titers, purified recombinant F1 (0.5 μg, the molar equivalent amount of protein present in 10¹¹ pu of AdLacZ-pIX/F1) with Alhydrogel (p < 0.0005, F1 unboosted vs. naive). (C) Survival, AdLacZ-pIX/F1. Six weeks after prime immunization with 10¹¹ pu of AdLacZ-pIX/F1, mice were challenged with a lethal dose of Yersinia pestis CO92, and survival of the animals was monitored for 14 days. Naive mice and mice that received AdLacZ (10¹¹ pu) intramuscularly were included as negative controls (p < 0.0007, AdLacZ-pIX/F1 vs. AdLacZ; p < 0.0001, AdLacZ-pIX/F1 plus boost vs. AdLacZ. (D) Survival, F1/Alhydrogel. Naive mice were used as negative controls (p = 0.4187, F1 vs. naive; p < 0.0001, F1 plus boost vs. naive).