IgG effector mechanisms are influenced by the attached Fc N
-glycans. Here we studied changes in IgG Fc N
-glycosylation upon vaccination of 10 Caucasian adults and 10 African children. To our knowledge, glycosylation of the constant region of human plasma IgG1 is restricted to N
-glycosylation at Asn297, and no literature reports on O
-glycosylation of the constant region are available. IgG Fc N
-glycosylation profiles were determined using a recently described fast nanoLC-ESI-MS method, which allows accurate registration of tryptic IgG1, IgG2 and IgG4 Fc N
-glycopeptides in a single analysis (24
). The relative expression levels were determined for a total of 46 IgG Fc N
-glycopeptides () which were assigned unambiguously on the basis of literature knowledge of IgG N
). From these data a set of IgG Fc N
-glycosylation features, namely fucosylation, galactosylation, sialylation, sialic acids per galactose, and the level of bisecting N
-acetylglucosamine were determined (24
There were no vaccine related longitudinal changes in glycosylation of total IgG1, IgG2, and IgG4. We did detect glycosylation changes of vaccine specific IgG1 in time. Active immunization with influenza or tetanus toxoid induced higher levels of galactosylation and sialylation and decreased the bisecting GlcNAc of antigen-directed IgG1. Interestingly, we observed an increase in the number of sialic acids per galactose upon vaccination which might indicate a differential regulation of β4-galactosyltransferase and sialyltransferase activities involved in IgG Fc N
-glycosylation during biosynthesis in B lymphocytes (9
). We did not observe significant changes in the level of fucosylation for total IgG and antigen-directed IgG1. No further changes in the glycosylation profiles were observed upon the second immunization of the Caucasians with influenza (day 21), which was possibly because of the large time difference (35 days) between the boost vaccination and sampling.
Our results are in contrast to the results of murine immunization studies: active immunization of specific pathogen free CBA/Ca mice with BSA causes a decrease in the galactosylation level for anti-BSA IgG (10
). In a murine serum nephritis model, immunization caused a drastic reduction of the IgG sialic acid content (4
). Upon repeated immunization with ovalbumin, increased levels of IgG fucosylation have been observed for male ICR mice (11
). Specific pathogen free mice transferred from a sterile to a conventional environment showed an initial increase in the total IgG galactose content up to day 17 after which it decreased (10
). This galactosylation effect on the level of total IgG is similar to our observation of an initial galactosylation increase on vaccine specific directed IgG1. However, specific pathogen free control mice remaining in the sterile environment revealed a similar galactosylation change, suggesting that the observed effect was caused by aging of the mice rather than due to infection. Murine and human IgG subclasses/isotypes are different in various respects including their glycosylation, as murine IgG Fc N
-glycans contain no bisecting GlcNAc and may carry N
-glycolylneuraminic acid which is not found on human IgG (32
). In addition, lectins expressed on murine effector cells differ from those expressed in humans (e.g.
SIGN-R1 an orthologue of the human DC-SIGN) (19
). Furthermore, murine glycoproteins and glycoproteins including IgG expressed in murine cell lines contain Galα1,3-Gal epitopes (35
). Hence, the study of specific glycosylation changes in murine models might not translate directly to the situation in humans.
IgG1 Fc N
-glycans containing a bisecting N
-acetylglucosamine have been shown to exhibit increased ADCC potency in in vitro
). The decrease in the level of bisecting N
-acetylglucosamine on antigen-directed IgG1 upon vaccination might, therefore, suggest a lower ADCC potency of the anti-vaccine IgG1. While the high level of IgG1 Fc galactosylation found in our study is likewise expected to result in rather weak interactions with activating Fc receptors and, consequently, ADCC (4
), high levels of Fc galactosylation have been found to lead to enhanced complement-dependent cytotoxicity (38
). Tetanus toxoid (40
) and influenza envelope glycoprotein (hemagglutinin and neuraminidase) (41
) vaccines elicit high neutralizing antibody responses which have been correlated to vaccine-induced protective immunity. Influenza vaccine induced effector functions by nonneutralizing antibodies have also been shown to be involved in influenza clearance (44
), and these effector functions are expected to be modulated by the glycosylation changes described here. The precise effector mechanisms involved in vaccine-mediated protection are far from clear and different mechanisms might apply for different viruses under different conditions (49
B lymphocytes may produce IgGs with distinct Fc N
-glycosylation profiles as is exemplified by the glycosylation differences between vaccine specific IgG1 and total serum/plasma IgG1. First steps toward elucidating the underlying regulatory IgG glycosylation mechanisms in B cells have recently been performed (9
). The glycosylation of IgG1 produced by B lymphocytes in vitro
is influenced by environmental factors (all-trans retinoic acid) and factors known to stimulate the innate (i.e.
CpG oligodeoxynucleotide) or adaptive (i.e.
interleukin 21) immune system (9
). Interestingly, the reported short-term increase in galactosylation and decrease in bisecting GlcNAc of IgG1 in oligodeoxynucleotide or interleukin 21 stimulated B cells is in agreement with our observations during vaccination of humans. Modern vaccines such as those used in this study often contain adjuvants to enhance the immunogenicity of subunit (microbe strains and purified proteins) and DNA vaccines. Adjuvants can modify the outcome of epitope presentation to the immune system by specific TH
2 polarization efficacy (50
). The observed IgG Fc N
-glycosylation changes, therefore, might be a result of a combined immune response toward the antigens and the adjuvant.
One may expect that prior to vaccination the individuals have had several encounters with cross reactive influenza strains via infections or previous vaccinations resulting in the resting state IgG glycosylation profile at day 0. Although glycosylation changes were observed within weeks after vaccination, IgG1 Fc glycosylation profiles obtained 9 months after influenza vaccination (determined for four African children, data not shown) were very similar to the profiles at day 0 and are likewise interpreted as resting state profiles.
Seasonal flu (influenza) vaccination usually precedes the encounter with the virus by weeks or months (prophylactic). Hence antibodies with high galactosylation and sialylation but with low incidence of bisecting GlcNAc are expected to be the ones involved in the defense against seasonal flu. Tetanus vaccination provides two scenarios as it is often performed directly after a wound (curative), and as preventive vaccination (prophylactic) which protects the individual 10–15 years. Our data indicate that dependent on the vaccination time point the infectious agent will encounter IgGs with different glycosylation profiles (acute, high galactosylation, high sialylation versus
resting state, low galactosylation, low sialylation) which might influence the antibody effector functions relevant in immunity. Interestingly, the observed switching points in the glycosylation features follow the antibody titers (data not shown) (21
In conclusion, analysis of different populations and races shed some light on natural effects of vaccination on antibody glycosylation profiles. Obviously, glycosylation patterns observed by us upon vaccination can not be easily explained from a teleological point of view, but it should be stressed that the regulatory aspects and functional implications of human IgG glycosylation features are still largely unknown, and that further research is required.