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1.  Construction and Immunogenicity of Recombinant Adenovirus Vaccines Expressing the HMW1, HMW2, or Hia Adhesion Protein of Nontypeable Haemophilus influenzae▿  
Clinical and Vaccine Immunology : CVI  2010;17(10):1567-1575.
The objective of the present study was to construct and assess the immunogenicity of recombinant adenovirus vectors expressing the HMW1, HMW2, or Hia protein of nontypeable Haemophilus influenzae (NTHi). These proteins are critical adhesins and potential protective antigens expressed by NTHi. Segments of the hmw1A and hmw2A structural genes that encode the distal one-half of mature HMW1 or HMW2 were cloned into the T7 expression vector pGEMEX-2. These constructs encoded stable HMW1 or HMW2 recombinant fusion protein that expresses B-cell epitopes common to most NTHi strains. A segment of the hia gene that encodes the surface-exposed portion of mature Hia was also cloned into pGEMEX-2. The resulting T7 gene 10 translational fusions were excised from the parent plasmids and cloned into the shuttle plasmid pDC316. Cotransfection of HEK 293 cells with the pDC316 derivatives and pBHGloxΔE1,3Cre resulted in the production of viral plaques from which recombinant adenoviruses expressing fusion proteins were recovered. Chinchillas immunized intraperitoneally with a single 108-PFU dose of either the HMW2 or Hia adenoviral construct developed high anti-HMW2 or anti-Hia serum antibody titers within 4 weeks of immunization. Chinchillas immunized intranasally with a single 107- to 109-PFU dose of the Hia adenoviral construct also developed high anti-Hia serum antibody titers within 8 weeks of immunization. Recombinant adenoviruses represent a promising system to induce mucosal and systemic immunity and protection against mucosal diseases such as otitis media. Recombinant adenoviruses expressing recombinant HMW1, HMW2, or Hia protein will be important new tools in NTHi vaccine development efforts.
doi:10.1128/CVI.00115-10
PMCID: PMC2952983  PMID: 20685934
2.  Antibodies Specific for the Hia Adhesion Proteins of Nontypeable Haemophilus influenzae Mediate Opsonophagocytic Activity▿  
The Hia autotransporter proteins are highly immunogenic surface adhesins expressed by nontypeable Haemophilus influenzae (NTHI). The objective of our study was to assess the opsonophagocytic activity of anti-Hia antibodies against homologous and heterologous NTHI. A segment of the hia gene that encodes a surface-exposed portion of the H. influenzae strain 11 Hia protein was cloned into a pGEMEX-2 expression vector. Escherichia coli JM101 was transformed with the resulting pGEMEX-Hia BstEII del recombinant plasmid, and recombinant fusion protein was recovered. An immune serum against recombinant GEMEX-Hia (rGEMEX-Hia)-mediated killing of the homologous NTHI strain 11 at a 1:160 titer and five heterologous Hia-expressing strains at titers of ≥1:40. Immune serum did not mediate killing of two Hia-knockout strains whose hia genes were inactivated but did mediate killing of one knockout strain at a high titer after the strain was transformed with a plasmid containing the hia gene. Immune serum did not mediate killing of HMW1/HMW2-expressing NTHI strains, which do not express the Hia adhesin. However, when two representative HMW1/HMW2-expressing strains were transformed with the plasmid containing the hia gene, they expressed abundant Hia and were susceptible to killing by the immune serum. Immune serum did not mediate killing of HMW1/HMW2-expressing strains transformed with the plasmid without the hia gene. Our results demonstrate that the Hia proteins of NTHI are targets of opsonophagocytic antibodies and that shared epitopes recognized by such antibodies are present on the Hia proteins of unrelated NTHI strains. These data argue for the continued investigation of the Hia proteins as vaccine candidates for the prevention of NTHI disease.
doi:10.1128/CVI.00090-09
PMCID: PMC2708409  PMID: 19474261
3.  Antibodies Specific for the High-Molecular-Weight Adhesion Proteins of Nontypeable Haemophilus influenzae Are Opsonophagocytic for both Homologous and Heterologous Strains▿  
Clinical and Vaccine Immunology  2006;13(12):1333-1342.
The HMW1/HMW2-like adhesion proteins of nontypeable Haemophilus influenzae (NTHI) are expressed by 75% of NTHI strains. Antibodies directed against these proteins are opsonophagocytic in vitro and are protective in an animal model of infection. The objective of the present study was to determine the opsonophagocytic activity of high-titer anti-HMW1/HMW2 immune sera against both homologous and heterologous NTHI strains. Chinchillas were immunized with purified HMW1/HMW2-like proteins from five prototype NTHI strains. Serum opsonophagocytic activity was monitored in an assay that uses a human promyelocytic cell line, HL-60, as the source of phagocytic cells. Preimmune sera did not demonstrate opsonophagocytic killing of any strains. In contrast, the immune sera demonstrated killing of the five homologous NTHI strains at titers ranging from 1:320 to 1:640. The immune sera also demonstrated killing of eight heterologous NTHI strains that express HMW1/HMW2-like proteins at titers ranging from 0 to 1:640. Killing of heterologous strains sometimes demonstrated a prozone phenomenon. None of the immune sera killed NTHI strains that did not express HMW1/HMW2-like proteins. Adsorption of immune sera with HMW1/HMW2-like proteins purified from either homologous or heterologous NTHI strains eliminated opsonophagocytic killing of homologous strains in most cases. These data demonstrate that antibodies produced following immunization with the HMW1/HMW2-like proteins are opsonophagocytic for both homologous and heterologous NTHI and strongly suggest that common epitopes recognized by functionally active antibodies exist on the HMW1/HMW2-like proteins of unrelated NTHI strains. The results argue for the continued investigation of the HMW1/HMW2-like proteins as potential vaccine candidates for the prevention of NTHI disease.
doi:10.1128/CVI.00221-06
PMCID: PMC1694446  PMID: 17021246
4.  Human Antibodies Specific for the High-Molecular-Weight Adhesion Proteins of Nontypeable Haemophilus influenzae Mediate Opsonophagocytic Activity  
Infection and Immunity  2003;71(12):6884-6891.
The HMW1- and HMW2-like adhesion proteins of nontypeable Haemophilus influenzae are expressed by 75% of these strains, and antibodies directed against these proteins are protective in animal models of infection. The purpose of the present study was to define the functional activity of human antibodies specific for these proteins in an in vitro complement-dependent opsonophagocytic assay. Human promyelocytic cell line HL-60 served as the source of phagocytic cells, and a commercial preparation of intravenous immunoglobulin (IVIG) served as the source of human antibodies. High-molecular-weight (HMW) proteins were purified from four prototype nontypeable H. influenzae strains and used to prepare solid-phase affinity columns. IVIG was adsorbed on each column to remove strain-specific anti-HMW antibodies and to allow recovery of affinity-purified anti-HMW antibody fractions. Unadsorbed IVIG killed each of the prototype strains at titers of 1:80 to 1:320. HMW-adsorbed sera demonstrated fourfold decreases in opsonophagocytic titer against the homologous strains compared to unadsorbed IVIG. Affinity-purified anti-HMW antibody preparations demonstrated opsonophagocytic titers of 1:20 to 1:80 against the respective homologous strains and opsonophagocytic titers as high as 1:80 against heterologous strains. None of the affinity-purified anti-HMW antibody preparations was opsonophagocytic for a representative nontypeable H. influenzae strain that did not express HMW1- or HMW2-like proteins. These data demonstrate that human antibodies specific for the HMW1/HMW2-like adhesion proteins of nontypeable H. influenzae are opsonophagocytic and that such antibodies recognize epitopes shared by the HMW proteins of unrelated nontypeable H. influenzae strains. These results argue for continued investigation of the HMW1/HMW2-like proteins as potential vaccine candidates for prevention of disease due to nontypeable H. influenzae.
doi:10.1128/IAI.71.12.6884-6891.2003
PMCID: PMC308909  PMID: 14638776

Results 1-4 (4)