Shielding Effect of Escherichia coli O-Antigen Polysaccharide on J5-Induced Cross-Reactive Antibodies

Abstract

Escherichia coli is the leading cause of severe mastitis in dairy farms. As E. coli mastitis is refractory to the hygienic control measures adapted to contagious mastitis, efficient vaccines are in demand. Existing mastitis vaccines, based on the use of killed rough E. coli J5 as the antigen, aim at inducing phagocytosis by neutrophils. We assessed the binding of J5-induced antibodies to isogenic rough and smooth strains along with a panel of mastitis-associated E. coli. Analysis by enzyme-linked immunosorbent assay revealed that antibodies to OmpA or killed J5 bind readily to rough E. coli but poorly to smooth strains. Flow cytometry analysis indicated that immunization with J5 induced antibodies that cross-reacted with rough E. coli strains but with only a small subpopulation of smooth strains. We identified type 1 fimbriae as the target of most antibodies cross-reacting with the smooth strains. These results suggest that the O-polysaccharide of lipopolysaccharide shields the outer membrane antigens and that only fiber antigens protruding at the bacterial surface can elicit antibodies reacting with mastitis-associated E. coli. We evaluated J5-induced antibodies in an opsonophagocytic killing assay with bovine neutrophils. J5 immune serum was not more efficient than preimmune serum, showing that immunization did not improve on the already high efficiency of naturally acquired antibodies to E. coli. In conclusion, it is unlikely that the efficiency of J5 vaccines is related to the induction of opsonic antibodies. Consequently, other research directions, such as cell-mediated immunity, should be explored to improve E. coli mastitis vaccines. IMPORTANCE Despite intensive research, mastitis remains an important disease in dairy cattle with a significant impact on animal welfare, use of antibiotics, and, in the end, the economy of dairy farms. Although vaccines available so far have shown limited efficacy against coliform mastitis, vaccination is considered one of the measures that could limit the consequences of mastitis. One reason for the lack of efficiency of current vaccines likely stems from the current evaluation of vaccines that relies mostly on measuring antibody production against vaccine antigens. This report clearly shows that vaccine-induced antibodies fail to bind to most mastitis-associated E. coli strains because of the presence of an O-antigen and, thus, do not allow for improved phagocytosis of pathogens. As a consequence, this report calls for revised criteria for the evaluation of vaccines and suggests that cell-mediated immunity should be targeted by new vaccinal strategies. More generally, these results could be extended to other vaccine development strategies targeting coliform bacteria.

Invited review: A critical appraisal of mastitis vaccines for dairy cows

“…This shielding has been confirmed with mastitis strains: immunization of cows with J5 induced cross-reactive antibodies to rough strains but these antibodies, except those directed at fimbriae, reacted very poorly with smooth strains and did not improve on the opsonic activity of preexisting natural antibodies (Figure 1) (Rainard et al, 2021). Absorption of J5 immune serum with smooth strains did not reduce the antibody reactivity with rough strains, demonstrating the efficient shielding by the O-antigen polysaccharide layer toward outer membrane antigens.…”

“…In accordance, immunization of rabbits with E. coli J5 did not augment the opsonization titer to smooth strains (van Dijk et al, 1981). Nevertheless, J5 bacterins induce cross-reactive antibodies, even though their specificity has not been characterized (Ziegler et al, 1973), except for the outer membrane protein OmpA and type 1 fimbriae (Rainard et al, 2021). The role played by these cross-reacting antibodies in protection against infection has been evaluated in vaccination and challenge experiments.…”

“…The specificity of the opsonic antibodies was not determined. It appeared that J5 vaccines elicit antibodies to molecules protruding from the O-antigen shield, such as type 1 fimbriae, which can bind to smooth strains (Rainard et al, 2021). Since a proportion of mastitis E. coli isolates have the genetic equipment to produce these structures (Lipman et al, 1995), and as fimbriae are supposed to contribute to adhesion to mammary epithelial cells (MECs), antibodies to fimbriae could play a role in vaccine-induced protection.…”

“…Since a proportion of mastitis E. coli isolates have the genetic equipment to produce these structures (Lipman et al, 1995), and as fimbriae are supposed to contribute to adhesion to mammary epithelial cells (MECs), antibodies to fimbriae could play a role in vaccine-induced protection. However, their opsonic activity has not been evidenced (Rainard et al, 2021).…”

Progress towards the Elusive Mastitis Vaccines

“…Moreover, the recognized epitopes may not be the right targets of effector activity. They can also be shielded by a bacterial capsule or other surface polysaccharides, as are the outer membrane proteins of E. coli mastitis isolates, which are beyond the reach of the antibodies induced by the rough E. coli J5 [146].…”