V. Cholerae O139 Lps-the Ctb Conjugate Vaccines

Cholera is an acutely dehydrating, watery diarrhoeal disease caused by intestinal infection with the gram-negative bacillus Vibrio cholerae. Human beings are natural hosts of Vibrio cholerae because of ingesting contaminated water or food by this bacterium. Severe cholera is characterized by profuse watery diarrhoea and vomiting, which leads within hours to hypovolemia, metabolic acidosis and potassium deficiency arising from the loss of fluid and electrolytes. Complications include renal failure, arterial occlusions, pulmonary edema, abortion in pregnant women and profound hypoglycemia and seizures in young children. Cholera epidemics are associated with case fatality rates exceeding 20% and led to tens of thousands of deaths. The epidemic is characterized by explosive, which means that it starts simμltaneously in several different locations and then spreads rapidly to the rest of the world, particularly in Africa, Asia and Latin America and other developing countries. Seven cholera pandemics have been recorded since the early nineteenth century. An effective way to control it is prophylactic immunization.It has been confirmed that lipopolysaccharide (LPS) viz. bacterial antigen (O- antigen) on the cell wall of Vibrio cholerae is not only an important virulence factor but also a protective antigen. LPS are, however, weak immunogens which induce mainly IgM antibodies and little, if any, memory response and booster immunization. And infants respond poorly to these antigens and are thus susceptible to infections from bacteria. Cholera toxin B (CTB) whicn is the non-toxic part of cholera toxin has good immunogenicity and can induce important anti-toxin antibodies. And CTB has been proved to be a good mucosal adjuvant which can induce mucosal immune response.In this research, LPS was covalently attached to CTB. This conjugation converted LPS to a T-cell dependent antigen which can induce an anamnestic response and booster immunization with class switching, and produce anti-vibrio antibodies. Besides,CTB itself can produce high titers of anti-toxin antibodies. These are the two aspects should be involved in anti-cholera vaccines.In this research, LPS was extraced and purificated from Vibrio cholerae O139 by using hot phenol water method. 1-cyanide-4-dimethyl amino pyridine tetrafluoroborate (CDAP) was ued to activate LPS and then the activated LPS was covalently coupled of CTB. This product was purificated by passing through the Superdex-200 gel filtration column purification. The final product was designated high purity LPS-CTB conjugates. Solutions of LPS, alone or as a conjugate, and rBS-WC vaccine were injected intraperitonealy into young mice, and LPS and CTB antibodies were measured by enzyme-linked immunosorbent assaying. The conjugates could induce not only high titer of anti-LPS and anti-CTB serum IgG and IgA( The titer of anti-LPS IgG and IgA antibodies were 1:970 and 1:1691,and the titer of anti-LPS antibodies both were as high as 1: 163840), but also induce secretory IgA antibodies which are extremely important in the cholera immunization. Furthermore, it induced booster immunization after repeated immunization. All these proved the LPS-CTB conjugates had good immunogenicity.This cholera vaccine will have a good prospect in cholera prevention.