The Intracerebral Infection of Mice with Hemophilus Influenzae as an Index of Strain Virulence and the Protective Value of Immune Serum

Review: Evidence against the hypothesis that antibodies to the inner core of lipopolysaccharides in antisera raised by immunization with enterobacterial deep-rough mutants confer broad-spectrum protection during Gram-negative bacterial sepsis

“…'z (We recognize the possibility that this discrepancy might simply reflect differences between protective activities of murine and rabbit antibodies.) 5. Broad-spectrum protection in granulocytopenic rabbits actively immunized with the R595 mutant was attributed to antibodies to the Re LPS even though comparable titers of polyclonal serotypespecific antibodies, which possess considerably greater protective activity, 12,15,16,27 were evoked by the immunization.…”

“…For example, Young and Stevens indicated that the protection afforded by R595 rabbit and canine antisera in mice challenged with E. coli was 'critically dependent' upon the concentration of hog gastric mucin administered concomitantly.&dquo;5 5 Sakulramrung and Dominque reported that no protection was afforded by high-titer J5 rabbit antiserum given to mice 1 h before challenge with 2 LD 50 of K. pneumoniae, E. coli 06, or E. coli 04, or even with K. pneumoniae that had been preincubated with the antiserum,5° confirming our and other investigators' negative findings.35-4' This antiserum, however, protected mice against challenge with viable E. coli 04 when sheep hemoglobin was administered concomitantly.5° Appelmelk et al demonstrated that J5 rabbit antisera could not protect normal mice against challenge with the parental smooth E. coli (again confirming the previous negative findings), but could protect if the mice were given mucin plus hemoglobin.2 1 Appelmelk et al hypothesized that immunocompromising agents are required to demonstrate protection by antisera to rough mutants because antibodies to core LPS epitopes are relatively ineffective compared with antibodies to 0-specific antigens; but these antibodies become protective against challenge with the smaller inocula used in the compromised host because of the greater amounts of antibodies per organism .2 However, we would stress that the same mechanism would also magnify the protective activity of 0-specific antibodies. As reviewed above, antisera to rough mutants often contain polyclonal increments in O-specific antibodies.…”

“…159 This capability, however, is not unique and indeed would be expected for lipid A-reactive antibodies since it involves pretreatment of rabbits with LPS (preparatory dose) and has been demonstrated with polyvalent rabbit antisera to purified lipid A. I70 While lipid A-reactive antibodies may protect rabbits against the dermal Shwartzman reaction, these antibodies appear incapable of directly neutralizing LPS,72,170 or On the basis of the above considerations, we believe that the broad-spectrum protective activity against lethality, from wild-type Gram-negative bacteria and their S-form LPS attributed to two lipid A-reactive MAbs, 8A1 and A6(H4C5),~s,i59 could be mediated by mechanisms other than broad-spectrum binding to, and subsequent neutralization of, S-form LPS. First, and most importantly, no precautions were specified for ensuring that the MAbs 8A1 and A6(H4C5) or their controls were free of contaminating endotoxin.87811,119 Ensuring that these MAbs were endotoxin-free would be essential in those studies in which these antibodies were given [2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18] h before challenge,&dquo;9 since nonspecific enhancement of host resistance to infection and to LPS occurs rapidly after exposure to trace amounts of LPS. 172,171 Ensuring that the control preparations are free of endotoxin contamination is essential when bacterial or LPS challenge is performed immediately after administration of the test preparations,87,88 since contamination of the control preparation with LPS can elevate mortality in the control group, making it appear that the J5 or R595 MAbs are protective.…”

“…For example, no studies used whole sera or Sephadex G-200 fractions of whole sera obtained from volunteers vaccinated with wild-type smooth Gram-negative organisms to evoke comparable local and systemic inflammatory and febrile responses without producing increments in antibodies to R595 epitopes. 5. The possibility that polyclonal serotype-specific antibodies, rather than antibodies to R595 LPS, in R595 antisera could account for protection was excluded on the basis that titers of specific antibodies 'did not increase materially' in human sera as assessed by HA assays.…”