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Selectively Split Whole Cell Vaccines

A whole-cell, cell-based technology, applied in the fields of immunology, bacteriology, and molecular genetics, can solve problems such as immunogenicity exploration

Active Publication Date: 2016-01-13
CHILDRENS MEDICAL CENT CORP +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Alternative strategies have used inactivated pneumococcal cells (presenting multiple serotype-independent antigens) as inexpensive vaccines, but the immunogenicity of these compositions has not been adequately explored

Method used

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  • Selectively Split Whole Cell Vaccines
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  • Selectively Split Whole Cell Vaccines

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0102] Example 1. Antigen preparation

[0103] Strain Rx1E in which the pneumolysin gene was replaced by a detoxified mutant PdT was provided to us by James Paton (University of Adelaide, Australia). Using the strategy described previously, the entire lytA genomic coding region was replaced with a Janus expression cassette tagged with the kanamycin resistance gene rpsL (Sung et al., Appl. Environ. Microbio. 139 (2006); van Ginkel et al., J. Immunol. , 165, 4778 (2000)). Briefly, three PCR amplification products were generated: (i) with primers LAD1 (CAAGGTATCCATCATTCC) (SEQ ID NO: 1) and LAD2 (CGC GGATCC The 1 kb fragment upstream of the lytA genomic region amplified by ACAGTAGAGCCAGATGGC (SEQ ID NO: 2); the BamHI site is underlined); (ii) with primer LAD3 (TTT GGGCCC GTTGCACGCCGACTTGAGG (SEQ ID NO:3); ApaI site is underlined) and LAD4 (CTTTGCTTCTCAGAATCTAGG) (SEQ ID NO:4) amplified 800bp fragment downstream of lytA; and (iii) primers DAM351 (with ApaI site) and DAM406 ( ...

Embodiment 2

[0108] Example 2. Immunization and challenge of mice

[0109] C57BL / 6J mice (Jackson Laboratories, Bar Harbor, Maine) were used in all experiments. Age 4-6 weeks at the time of first immunization. Intranasal (i.n) immunization (a step that does not place the immunogen into the lungs) by atraumatically instilling 10 μL of saline, adjuvant alone, or adjuvant mixed with the specific antigen into non-anesthetized mice ; a second immunization was performed 1 week later. respectively by mixing with 1%NaHCO 3 Oral or sublingual immunizations were performed by placing 30 μL of the vaccine mixed with 30% sucrose on the oral surface or by placing 5 μL of the vaccine in the same diluent under the tongue. Oral or sublingual immunizations were given three times a week, whereas intranasal immunizations were given only twice.

[0110] WCC was used for transdermal immunoassay (TCI) experiments. WCCs were rehydrated in water containing 0.1% Zwittergent3-14 (Calbiochem, Gibbstown, NJ) and ...

Embodiment 3

[0114] Example 3. Rabbit Immunization and Toxicology Studies

[0115] All rabbit immunizations were performed at MPI (Mattawan, MI). On days 1, 15, 29 and 43, female New Zealand White rabbits in groups of three were given 0.5 ml of the following injections by intramuscular injection: saline; Al(OH alone ) 3 (contains 0.6 mg Al); doses of 50 μg, 500 μg or 5000 μg of Al(OH) 3 Adsorbed WCB or whole-cell diphtheria-tetanus-pertussis whole-cell (DTwP) vaccine (clinical product from Institute Butantan). Sera were obtained before each immunization and at the moribund exsanguination on day 45 and sent frozen to Children's Hospital Boston for antibody determination. For all animals, observations were made periodically for morbidity, mortality, clinical signs, body temperature, and food and water consumption. Dermal irritation scores were evaluated before each administration, and after each administration (except the last administration) were evaluated daily for three consecutive da...

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Abstract

The present invention provides immunogenic compositions having an inactivated, whole-cell S. pneumoniae, multiplex immunity-inducing fraction and methods of making immunogenic compositions by The immunogenic composition is prepared by selectively lysing a whole-cell bacterial preparation in such a way that the soluble fraction that primarily induces an antibody response and the cellular fraction that primarily induces an antibody-independent response are retained in the immunogenic composition.

Description

[0001] Cross References to Related Applications [0002] This application is based on 35 U.S.C. § 119(e), U.S. Code, claiming U.S. Provisional Patent Application No. 61 / 250,348, filed October 9, 2009, and U.S. Provisional Patent Application No. 61 / 380,429, filed September 7, 2010 The priority of the two is incorporated herein by reference in their entirety. [0003] governmental support [0004] This invention was made with US Government support under R01AI066013, AI067737-01, and AI51526-01 awarded by the National Institutes of Health, which holds certain rights in this invention. technical field [0005] The present invention relates to molecular genetics, immunology and bacteriology. In particular, embodiments of the invention provide whole cell immunogenic formulations that confer a synergistic antibody-mediated and T-lymphocyte-mediated immune response. In the case of pneumococci, this whole-cell immunogenic preparation elicited synergistic antibody-mediated and T-lymp...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K39/395A61K39/09A61K39/02A61P31/00
CPCA61K39/092A61K2039/521A61K2039/543A61K2039/55505A61K2039/55544A61P31/00A61P31/04A61P37/04
Inventor 理查德·马利波特·安德森陆英杰马克·奥尔德森乔治·A·罗伯逊让-弗朗索瓦斯·卢西恩·迈松纳夫安德烈亚·马里亚·泰特瓦尔德里·德奥利韦拉·迪亚斯维维亚娜·迈蒙里·贡萨尔维斯
Owner CHILDRENS MEDICAL CENT CORP
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