Construction and Immunogenicity of Recombinant Adenovirus Vaccines Expressing the HMW1, HMW2, or Hia Adhesion Protein of Nontypeable Haemophilus influenzae

ABSTRACT 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.

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Multiple Consecutive Lavage Samplings Reveal Greater Burden of Disease and Provide Direct Access to the Nontypeable Haemophilus influenzae Biofilm in Experimental Otitis Media

Infection and Immunity ◽

10.1128/iai.00318-07 ◽

2007 ◽

Vol 75 (8) ◽

pp. 4158-4172 ◽

Cited By ~ 21

Author(s):

Magali Leroy ◽

Howard Cabral ◽

Marisol Figueira ◽

Valérie Bouchet ◽

Heather Huot ◽

...

Keyword(s):

Gene Expression ◽

Haemophilus Influenzae ◽

Otitis Media ◽

Vaccine Development ◽

Ex Vivo ◽

Direct Analysis ◽

Direct Access ◽

Nontypeable Haemophilus Influenzae ◽

Nasopharyngeal Colonization

ABSTRACT The typically recovered quantity of nontypeable Haemophilus influenzae (NTHi) bacteria in an ex vivo middle ear (ME) aspirate from the chinchilla model of experimental otitis media is insufficient for direct analysis of gene expression by microarray or of lipopolysaccharide glycoforms by mass spectrometry. This prompted us to investigate a strategy of multiple consecutive lavage samplings to increase ex vivo bacterial recovery. As multiple consecutive lavage samples significantly increased the total number of bacterial CFU collected during nasopharyngeal colonization or ME infection, this led us to evaluate whether bacteria sequentially acquired from consecutive lavages were similar. Comparative observation of complete ex vivo sample series by microscopy initially revealed ME inflammatory fluid consisting solely of planktonic-phase NTHi. In contrast, subsequent lavage samplings of the same infected ear revealed the existence of bacteria in two additional growth states, filamentous and biofilm encased. Gene expression analysis of such ex vivo samples was in accord with different bacterial growth phases in sequential lavage specimens. The existence of morphologically distinct NTHi subpopulations with varying levels of gene expression indicates that the pooling of specimens requires caution until methods for their separation are developed. This study based on multiple consecutive lavages is consistent with prior reports that NTHi forms a biofilm in vivo, describes the means to directly acquire ex vivo biofilm samples without sacrificing the animal, and has broad applicability for a study of mucosal infections. Moreover, this approach revealed that the actual burden of bacteria in experimental otitis media is significantly greater than was previously reported. Such findings may have direct implications for antibiotic treatment and vaccine development against NTHi.

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Detection and characterization of pediatric serum antibody to the OMP P5-homologous adhesin of nontypeable Haemophilus influenzae during acute otitis media

Vaccine ◽

10.1016/s0264-410x(02)00306-7 ◽

2002 ◽

Vol 20 (29-30) ◽

pp. 3590-3597 ◽

Cited By ~ 15

Author(s):

Laura A Novotny ◽

Michael E Pichichero ◽

Philippe A Denoël ◽

Cecil Neyt ◽

Sylvie Vanderschrick ◽

...

Keyword(s):

Haemophilus Influenzae ◽

Otitis Media ◽

Serum Antibody ◽

Acute Otitis Media ◽

Nontypeable Haemophilus Influenzae

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Production of Nontypeable Haemophilus influenzae HtrA by Recombinant Bordetella pertussis with the Use of Filamentous Hemagglutinin as a Carrier

Infection and Immunity ◽

10.1128/iai.73.7.4295-4301.2005 ◽

2005 ◽

Vol 73 (7) ◽

pp. 4295-4301 ◽

Cited By ~ 12

Author(s):

Sylvie Alonso ◽

Eve Willery ◽

Genevieve Renauld-Mongénie ◽

Camille Locht

Keyword(s):

Immune Responses ◽

Haemophilus Influenzae ◽

Bordetella Pertussis ◽

Whooping Cough ◽

Etiologic Agent ◽

Nontypeable Haemophilus Influenzae ◽

Bacterial Surface ◽

Filamentous Hemagglutinin ◽

Bacterial Vector ◽

Antibody Titers

ABSTRACT Bordetella pertussis, the etiologic agent of whooping cough, is a highly infectious human pathogen capable of inducing mucosal and systemic immune responses upon a single intranasal administration. In an attenuated, pertussis toxin (PTX)-deficient recombinant form, it may therefore constitute an efficient bacterial vector that is particularly well adapted for the delivery of heterologous antigens to the respiratory mucosa. Filamentous hemagglutinin (FHA) has been used as a carrier to present foreign antigens at the bacterial surface, thereby inducing local, systemic, and protective immune responses to these antigens in mice. Both full-length and truncated (Fha44) forms of FHA have been used for antigen presentation. To investigate the effect of the carrier (FHA or Fha44) on antibody responses to passenger antigens, we genetically fused the HtrA protein of nontypeable Haemophilus influenzae to either FHA form. The fha-htrA and Fha44 gene-htrA hybrids were expressed as single copies inserted into the chromosome of PTX-deficient B. pertussis. Both chimeras were secreted into the culture supernatants of the recombinant strains and were recognized by anti-FHA and anti-HtrA antibodies. Intranasal infection with the strain producing the FHA-HtrA hybrid led to significantly higher anti-HtrA and anti-FHA antibody titers than those obtained in mice infected with the Fha44-HtrA-producing strain. Interestingly, the B. pertussis strain producing the Fha44-HtrA chimera colonized the mouse lungs more efficiently than the parental, Fha44-producing strain and gave rise to higher anti-FHA antibody titers than those induced by the parental strain.

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Design and assessment of TRAP-CSP fusion antigens as effective malaria vaccines

10.1101/613653 ◽

2019 ◽

Author(s):

Chafen Lu ◽

Gaojie Song ◽

Kristin Beale ◽

Jiabin Yan ◽

Emma Garst ◽

...

Keyword(s):

Malaria Vaccine ◽

Vaccine Development ◽

Antibody Responses ◽

Effective Vaccine ◽

Repeat Region ◽

Partial Protection ◽

Adhesion Protein ◽

Malaria Vaccines ◽

Multi Stage ◽

Antibody Titers

AbstractThe circumsporozoite protein (CSP) and thrombospondin-related adhesion protein (TRAP) are major targets for pre-erythrocytic malaria vaccine development. However, the most advanced CSP-based vaccine RTS,S provides only partial protection, highlighting the need for innovative approaches for vaccine design and development. Here we design and characterize TRAP-CSP fusion antigens, and evaluate their immunogenicity and protection against malaria infection. TRAP N-terminal folded domains were fused to CSP C-terminal fragments consisting of the C-terminal αTSR domain with or without the intervening repeat region. Homogenous, monomeric and properly folded fusion proteins were purified from mammalian transfectants. Notably, fusion improved expression of chimeras relative to the TRAP or CSP components alone. Immunization of BALB/c mice with the P. berghei TRAP-CSP fusion antigens formulated in AddaVax adjuvant elicited antigen-specific antibody responses. Remarkably, fusion antigens containing the CSP repeat region conferred complete sterile protection against P. berghei sporozoite challenge, and furthermore, mice that survived the challenge were completely protected from re-challenge 16 weeks after the first challenge. In contrast, fusion antigens lacking the CSP repeat region were less effective, indicating that the CSP repeat region provided enhanced protection, which correlated with higher antibody titers elicited by fusion antigens containing the CSP repeat region. In addition, we demonstrated that N-linked glycans had no significant effect on antibody elicitation or protection. Our results show that TRAP-CSP fusion antigens could be highly effective vaccine candidates. Our approach provides a platform for designing multi-antigen/multi-stage fusion antigens as next generation more effective malaria vaccines.

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Qualification of the Hemagglutination Inhibition Assay in Support of Pandemic Influenza Vaccine Licensure

Clinical and Vaccine Immunology ◽

10.1128/cvi.00368-08 ◽

2009 ◽

Vol 16 (4) ◽

pp. 558-566 ◽

Cited By ~ 55

Author(s):

Diana L. Noah ◽

Heather Hill ◽

David Hines ◽

E. Lucile White ◽

Mark C. Wolff

Keyword(s):

Pandemic Influenza ◽

Hemagglutination Inhibition ◽

Vaccine Development ◽

Serum Antibody ◽

Specific Reference ◽

Intermediate Precision ◽

Highly Pathogenic ◽

Regulatory Guidance ◽

H5n1 Influenza ◽

Antibody Titers

ABSTRACT Continued outbreaks of highly pathogenic avian influenza over the past decade have spurred global efforts to develop antivirals and vaccines. As part of vaccine development, standard methods are needed for determining serum antibody titers in response to vaccination. Hemagglutination inhibition (HAI) assays are appropriate for assessing the immunogenicity of pandemic influenza vaccines in support of license approval. We demonstrate that a rigorous qualification of the HAI assay for H5N1 influenza virus, evaluating for precision, intermediate precision, linearity, range, specificity, and robustness, satisfies the intent of regulatory guidance for assay validation despite the lack of availability of specific reference standard antigens and antisera.

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Human Adenovirus Serotypes Efficiently Transducing HEK293 Cells: An In Vitro Propagation of HAdv

International Journal for Research in Applied Sciences and Biotechnology ◽

10.31033/ijrasb.8.5.3 ◽

2021 ◽

Vol 8 (5) ◽

Author(s):

Mr. Utkalendu Suvendusekhar Samantaray ◽

Ms. Piyanki Santra

Keyword(s):

Vaccine Development ◽

Recombinant Adenovirus ◽

Scale Up ◽

Human Adenovirus ◽

Adenoviral Vectors ◽

Hek293 Cells ◽

Target Cells ◽

Hek 293 ◽

Mammalian Cell Lines

Generally, a gene which is inserted directly into a cell does not operate independently. Instead, the transmission of the gene is genetically modified by a biological messenger called a vector, consists of a transgene and a large DNA sequence as a backbone. Since they can deliver the new gene by infecting the cell, such viruses are also used as vectors. The adenovirus is a non-enveloped virus that can be tailored to transfer DNA to target cells, and it has sparked a lot of interest in the field, particularly in clinical trial therapy techniques. For the new age production of COVID-19 vaccine, development of different mammalian cell lines like HEK293 (most reliable growth and prosperity for transfection) and recombinant adenoviral vectors have become the first priority for biopharmaceutical giants and globally approved vaccine manufacturers to scale up their vaccine production. Adenoviruses have an icosahedral shape, with a protein coat encasing the viral double-stranded DNA genome. Because the adenovirus genome is relatively small, it's a good candidate for insertion of foreign DNA. The adenovirus E1A gene is deleted, and the virus loses its capacity to replicate. This ability can be restored during cell culture propagation by employing cells that produce the E1A protein, for example. Hence, in this mini research, I have shared an overview of the propagation of adenoviral vectors, i.e. recombinant adenovirus SARS CoV-2 vector in HEK-293 cell suspension culture.

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