Immunoglobulin G Subclass Profile of Antimeasles Response in Vaccinated Children and in Adults with Measles History

The aim of this study was to investigate measles-specific immunoglobulinG (IgG) subclass profile in vaccinated children and in adultswith natural infection. Serum samples were collected beforeand 30 days after vaccination. The sera from 51 late convalescentadults and seven adults with natural measles infection at the12th day after onset of rash have been also investigated. MeaslesIgG antibodies and specific IgG subclasses were tested by enzyme-linkedimmunosorbent techniques. In children younger than 3 years,the predominant subclass was IgG3, which contributed, on average,63.3% of the total IgG response. The contributions of specificIgG1 and IgG4 to the total IgG antimeasles response were lower(19.9% and 16.8%, respectively), whereas IgG2 was not found.In contrast, in the group of children older than 4 years, justIgG2 was a predominant subclass; it contributed 42.6% of thetotal IgG response. Other subclasses were also present but thecontribution was much lower. In adult volunteers with measleshistory, IgG2 was a predominant subclass of total IgG. Thus,in early convalescence IgG2 contributed 62% of the total IgGresponse, whereas in late convalescence the contribution waslower (41.4%). There were no visible differences in IgG subclasscomposition between subjects with natural infection and vaccinatedchildren except those below 3 years of age. The humoral immuneresponse of such subjects is immature and the IgG2 subclassof virus-specific antibodies has not been revealed in the sera.

INTRODUCTION

Top

Introduction

The antibiotic era is characterized by a shift of the humaninfection spectrum in favor of viral pathogens. Unfortunately,the number of antivirus agents is limited and vaccination remainsthe main route of virus infection control. In particular, measleshas been targeted for global eradication by the World HealthOrganization's Expanded Program of Immunization (1, 2). Despitethe wide use of attenuated virus vaccines, much is still unclear.

It is well known that the antibody response to viral antigensplays a critical role in removing free viral particles fromcirculation in the bloodstream and in limiting virus spreadin the host (18). Moreover, it was shown that the most effectivehumoral response to many viral protein antigens is providedby neutralizing antibodies of the immunoglobulin G (IgG) class(14). This class of immunoglobulins consists of four subclasses,each encoded by a separate C-region gene and endowed with uniquebiological functions that are important for an efficient humoralresponse to a given pathogen. Recently it was demonstrated thatantibody responses to viral protein antigens mainly are restrictedto IgG1, IgG3, or both (3, 5, 6, 9); IgG2 generally is stimulatedby carbohydrate antigens (12, 15), whereas IgG4 most likelyreflects chronic antigen stimulation (13). Therefore, the monitoringof specific IgG subclass profiles after vaccination, comparedwith natural virus infection, may give an insight into the mechanismsthat drive antibody production in both conditions.

The specific antimeasles IgG1, IgG2, IgG3, and IgG4 subclassresponse was measured with immunofluorescent method by bindingof IgG antibodies with Vero cells infected by the measles virusvaccine strain (8) or with an enzyme-linked immunosorbent assay(10). The first allows us to receive qualitative results only.On the other hand, standardization of solid-phase methods fordetermination of subclass composition of antiviral antibodies,including antimeasles antibodies, has been difficult becauseproperly standardized isotype-specific reagents and a standardserum with assigned weight-based units of different subclasseshave been missing. In this work we succeeded partly in resolvingthis problem using a collection of commercially available peroxidase-linkedmonoclonal antibodies against various IgG subclasses. A singlestandard serum was provided to compare the data obtained indifferent experiments.

The present study was undertaken to point out the specific antimeaslesIgG1, IgG2, IgG3, and IgG4 subclass response patterns elicitedafter vaccination or during natural infection.

MATERIALS AND METHODS

Top

Materials and Methods

Study population. Serum samples were collected from 30 children (12 girls and18 boys; median age, 1.39 years; range, 1 to 3 years) and 10children (five girls and five boys; median age, 4.78 years;range, 4 to 6 years) before and 30 days after vaccination, respectively,with a trivalent live attenuated measles, mumps, and rubellavaccine, Priorix (GlaxoSmithKline, Belgium).

Serum samples were also collected from 51 late-convalescentadults (more than 10 years after measles infection) and sevenadults with natural measles infection at the 12th day afterthe onset of rash. The serum collection was randomized. Serumsamples were stored at –20°C and used within 100 days.All children were seronegative before vaccination. Seroconversion(appearance of specific IgM and IgG antibodies) was reachedin 97.2% cases within 1 month. IgM measles antibodies have beenfound in the sera of all adults with early infection. The adultvolunteers also demonstrated IgG measles antibodies. Informedconsent was obtained from the parents and volunteers. The studywas approved by the Ethics Committee of the G. N. GabrichevskyInstitute of Epidemiology and Microbiology.

Assays. Measles IgM and IgG antibodies were tested by enzyme-linkedimmunosorbent techniques with a commercial kit (Human, Germany).Specific IgG subclasses were measured by an enzyme-linked immunosorbentassay (ELISA) standardized according to the general principlesof solid-phase immunoassay; the detailed protocol has been describedelsewhere (16). Briefly, we used measles antigen-coated 96-wellmicroplates from the ELISA test for the detection of IgG antibodiesto measles virus in human serum REF 51206 (Human, Germany).Serum samples were added at a dilution of 1:50 and then incubatedfor 30 min at 37°C.

During preliminary experiments we used various serum dilutions(1:20; 1:50; 1:100; and 1:500). The dilution of 1:50 showeda lack of nonspecific reactions and at the same time revealedboth low and high IgG concentrations in the sample. After washingwe added peroxidase-linked anti-IgG1 (clone 10G/2C11), IgG2(clone 23G/3C7), IgG3 (clone 22G/5G12), or IgG4 (clone 20G/5C7)monoclonal antibodies (Polygnost, St. Petersburg, Russia) ata concentration of 1 µg/ml according to the recommendationsinstead of anti-IgG conjugate belonging to the commercial kit.The plates were incubated at 37°C for 30 min. After washing,color reagent solution (tetramethylbenzidine) was added. Plateswere finally read at 450 nm on a microELISA reader. For comparingthe results of different experiments, a standard serum containingfour IgG subclasses of antimeasles antibodies has been used.A negative and positive control sera from the commercially kitfor the detection of anti-measles IgG antibodies have been usedas an additional control.

Statistical analysis. The differences in the frequency of specific subclasses wereanalyzed by the ${chi}$ ² test. The t test was employed to compare themeans of two groups. P values of <0.05 were considered significant.

RESULTS

Top

Results

In the group of children younger than 3 years, the predominantsubclass was IgG3. It was present in 100% of serum samples andcontributed, on average, 63.3% of the total IgG response. Thefrequency and contributions of specific IgG1 (72.4% and 19.9%,respectively) and IgG4 (65.5% and 16.8%, respectively) to thetotal IgG antimeasles response were low, whereas IgG2 was notfound (Fig. 1).

View larger version (34K):
[in this window]
[in a new window]
FIG. 1. Subclass profile of antimeasles IgG antibodies in children vaccinated with the trivalent live attenuated measles, mumps, and rubella vaccine Priorix (30 children were younger than 3 years and 10 children were older than 3 years). Serum samples were collected 30 days after vaccination. The data are presented as percent distribution of IgG subclasses in the total measles IgG response. Results are expressed as mean ± standard error of the mean.

In contrast, in older children (4 years and older) the predominantsubclass was IgG2. It was present in 90% of serum samples andcontributed 42.6% of total IgG response. Other subclasses werealso present but their contribution was much lower and averagedfor IgG1, IgG3, and IgG4 it was 12.0, 22.1, and 23.3%, respectively(Fig. 1). Significant difference was observed in antimeaslesIgG subclass distribution between the two groups of children(P < 0.001).

The subclass profile of antimeasles IgG antibodies of adultvolunteers with measles history is presented in Fig. 2. In thesesubjects as in vaccinated children older than 3 years, IgG2was the predominant subclass of total IgG. Thus, in early convalescence,IgG2 contributed 62% of the total IgG response, whereas in lateconvalescence, the contribution was lower (41.4%). Other subclasseswere also present. There were no significant differences betweenvarious IgG subclasses revealed in early and late measles convalescence.

View larger version (31K):
[in this window]
[in a new window]
FIG. 2. Subclass profile of antimeasles IgG antibodies in early (seven adults) and late (51 adults) measles convalescence. See also the legend to Fig. 1.

DISCUSSION

Top

Discussion

In the present study, the distribution of measles-specific antibodiesamong the four IgG subclasses was investigated in vaccinatedchildren and adults with natural infection (convalescent phase)or with long-lasting humoral immunity to measles. A predominantsubclass in adult subjects and in vaccinated children aged 4years or older was IgG2. On the contrary, in vaccinated childrenyounger than 3 years there were no antibodies belonging to theIgG2 subclass. It is very difficult to compare our data withthe results of previous investigations due to differences inthe experimental design and/or methodology. Nevertheless, itmay establish the fact that all four isotypes are present inthe sera of measles patients during the acute and memory phasesof the immune response (see Fig. 2). Hence, there is no reasonto consider that the antibody response to viral antigens isrestricted to any IgG subclass. Such a conclusion is in accordancewith the results obtained in other investigations (3, 6, 7,13).

In our opinion, the fact that IgG2 antibodies were lacking invaccinated children younger than 3 years is important but notsurprising. Similar results were obtained in children vaccinatedagainst hepatitis B virus. Thus, the children below 5 yearsof age responded mainly with IgG1 and IgG3 subclasses, whereasin older children anti-HBs antibodies belonging to all fourIgG subclasses have been revealed (6). The data correspond withwidespread opinions about the much slower maturation of theIgG2 subclass in human (4, 11, 17). In particular, it has beenshown that IgG1 and IgG3 levels increase rapidly, reaching themean adult values by 1.5 to 2 years of age, whereas the amountof IgG2 remains lower than 50% of the adult value, having notyet reached the normal concentration at the age of 9 to 12 years.

In conclusion, we failed to demonstrate visible differencesin IgG subclass composition between subjects with natural infectionand vaccinated children except those below 3 years of age. Itmay be postulated that the IgG subclass profile in measles-vaccinatedsubjects depends on age. The humoral immune response of childrenyounger than 3 years is immature, and the IgG2 subclass of virus-specificantibodies has not been revealed in the sera.

ACKNOWLEDGMENTS

We thank Andrei Toptygin for helpful discussions.

FOOTNOTES

* Corresponding author. Mailing address: G. N. Gabrichevsky Institute of Epidemiology and Microbiology, 10 Admiral Makarov Street, Moscow, 125212, Russia. Phone: 7-095-1118568. Fax: 7-095-4521830. E-mail: toptyginaanna{at}rambler.ru.

REFERENCES

Top