In the present study we have employed a validated ELISA for the detection and quantitation of serum antisqualene antibodies to show that immunization with vaccines containing the MF59 emulsion adjuvant does not induce antisqualene IgM or IgG antibodies. Instead, antisqualene antibodies were detected very frequently at low titers in sera from healthy individuals that had never received any vaccine containing squalene.
Our study confirms that squalene is very poorly immunogenic. Indeed, in studies carried out by Matyas et al. (12
), antibodies against squalene were elicited in mice only when squalene was formulated within liposomes containing endotoxin (lipid A). These data are in agreement with those previously reported showing that antibodies against cholesterol could be induced after immunization of mice with cholesterol-loaded liposomes containing lipid A (18
A clear finding of our study is that the vast majority of healthy adults have antibodies to squalene circulating in their sera. It is of note that these antibodies were found in individuals from various geographical areas, such as the United States, western Europe, and eastern Europe, which to our knowledge had never received vaccines or other pharmacological treatments containing squalene. The statistically significant difference found among the three cohorts studied here cannot be ascribed to vaccinations, since these people were never vaccinated previously with vaccines containing MF59. In some cohorts, the frequency of individuals with detectable levels of antisqualene antibodies was as high as 100%. This finding is reminiscent of similar findings reported by Alving et al. (2
), who showed that virtually all normal human sera contain naturally occurring antibodies against cholesterol. One may speculate that these naturally occurring antisqualene antibodies could behave as an immunomodulating mechanism for regulation of LDL and VLDL metabolism, as proposed for anticholesterol antibodies (3
), since LDL and VLDL transport both squalene and cholesterol in the bloodstream (13
). The hypothetical role of naturally occurring antisqualene antibodies still needs formal demonstration.
Due to its natural occurrence, squalene was used as the oil phase in the design of new adjuvant preparations, such as MF59. MF59 emulsion is used as an adjuvant for an influenza subunit vaccine licensed since 1997 in various countries in and outside Europe (16
). More than 20 million doses of this influenza vaccine with the MF59 adjuvant have been distributed with an excellent safety profile. In addition, MF59 has been tested and is being tested in clinical trials as an adjuvant for several new vaccines for various age groups and has always been associated with a good clinical tolerability (16
The interest in antisqualene antibodies was raised by reports claiming that these antibodies were detected in the sera of military personnel with the so-called Gulf War syndrome (4
). These reports were criticized on technical grounds, since the assay employed (Western blotting) was not validated and lacked any controls (1
). In addition, these claims were considered inconclusive by the Institute of Medicine (7
). Using a validated ELISA, we have shown formally that no increases in antisqualene antibodies over and above preexisting levels are observed following vaccination with influenza vaccine with the MF59 adjuvant, either at 1 month or at 6 months postvaccination. Interestingly enough, antisqualene antibodies were found at identical titers in the sera of control subjects that had been immunized with an influenza split vaccine without adjuvant.
In conclusion, we have shown that antisqualene antibodies of both IgG and IgM isotypes are detected very frequently in the sera of healthy adult individuals of different ages and from various geographical areas. Additionally, we have shown that vaccines with the squalene-containing MF59 adjuvant emulsion do not induce any increase either in the titer or in the proportion of subjects with antibodies against squalene. These data add to the safety profile of the MF59 adjuvant emulsion, already shown by extensive clinical trials and with the regular use of the licensed vaccine with the MF59 adjuvant in routine influenza immunization practices.