Cesare Belloni

Family study of non-responsiveness to hepatitis B vaccine confirms the importance of HLA class III C4A locus.

Non-responsiveness to hepatitis B virus (HBV) vaccine in adults is strongly associated with HLA-C4AQ0,DRB1*0301,DQB1*02 haplotype. This association was also demonstrated in neonates who failed to mount a humoral response to challenge with HBV vaccine. About 4% of vaccinated newborns do not reach a protective antibody level (⩾10 mIU/ml) at seroconversion and 0.4% is a non-responder even after receiving a fourth dose of vaccine (true non-responders (TNR)); while 3.6% achieved an antibody level ⩾10 mIU/ml (slow responders (SR)) only when reboostered with the fourth dose. In the present study we extend the vaccination and HLA typing to 91 family members of probands to understand better the possible parent-to-child transmission of this trait. A transmission disequilibrium test (TDT), performed in 27 families, showed that the C4AQ0 allele was almost always transmitted to probands, both TNRs and SRs. Although not statistically significant, the highest LOD score was obtained with C4A locus: 1.58. These results suggest the presence of a region regulating immune response against HBV vaccination near to or coincident with the C4A locus.

Immunogenicity of hepatitis B vaccine in term and preterm infants

Some studies have suggested that decreased seroconversion rates might be found in premature infants with low birthweight (< 2000 g) following administration of hepatitis B vaccine at birth. The aim of the present investigation was to evaluate possible differences in seropositive rates between full‐term and preterm infants after primary vaccination, in particular when gestational age or birthweight is very low. Two‐thousand and nine neonates born to HBs Ag‐negative mothers were vaccinated with 10 μg of recombinant hepatitis B virus (HBV) vaccine, from May 1991 to October 1994. Children with infections, congenital malformations or serious illnesses were excluded. HBV vaccine was administered intramuscularly, on the fourth day of life and again at 1 and 6 months of age. A 1‐ml blood sample was drawn from each infant 1 month after the third vaccine dose for determination of the level of anti‐HBs antibody. The response to HBV vaccination was evaluated in 241 preterm (gestational age < 38 weeks) infants and 1727 term neonates. No statistical difference was observed in the distribution of anti‐HBs antibody level, either between preterm infants (< 38 weeks) and newborns of normal gestational age, or between low birthweight (< 2500 g) and normal weight infants. The results suggest that preterm and low birthweight infants (< 2500 g) respond to HBV vaccine in the same measure as normal‐term infants.

Neonatal B lymphocyte subpopulations and method of delivery

We studied four groups of healthy term newborn infants: (1) 11 infants born by vaginal delivery; (2) 11 infants born by elective cesarean section; (3) 10 infants born by emergency cesarean section with labor, and (4) 10 infants born by complicated vaginal delivery. Total and differential leukocyte counts, cortisol blood level, and B lymphocyte subpopulations (SmIg, sIgD, sIgM, CD19, CD20, CD21, CD23) were evaluated in cord blood samples from the four infant groups. Furthermore, the Pentothal blood level was measured in infants born by elective cesarean section and in their mothers at delivery. Higher total and differential leukocyte counts and cortisol blood levels were observed in group 1 and 4 infants as compared with group 2 and 3 infants. A significant correlation was observed between cortisol blood level and leukocyte counts. The percentages of positivity to cell surface markers of B lymphocyte subpopulations were significantly higher in infants born by elective cesarean section. A negative significant correlation of thiopentone with sIgM and CD21 was observed. These data indicate a significant influence of method of delivery and of thiopentone on B lymphocyte subpopulations.

Early immunisation with hepatitis B vaccine: a five-year study.

We evaluated the response to the recombinant Hepatitis B vaccine using an accelerated schedule versus the traditional schedule by studying the immunologic memory induced in 200 children with HBs-Ag negative mothers. At seroconversion, the traditional schedule presented a higher percentage of children with serum HBs-Ag concentrations over 100 mIU/ml than the accelerated schedule. After five years this difference was no longer statistically significant and children who presented anti-HBsAg concentrations below 10 mUI/ml received an additional booster dose which stimulated the antibody concentration to exceed 100 mIU/ml in all cases. Recombinant HBV vaccine induced better long term immunologic memory when it was administered earlier.

Revaccination against hepatitis B virus of non-responding and low-responding infants immunised at birth. A parallel evaluation of rubella and tetanus vaccine

The aim of the present study was to identify the true extent of the non-responsiveness in infants born from HBsAg-negative mothers, vaccinated against Hepatitis B Virus (HBV) at birth. Sixty-four non- and low-responding infants, selected from an initial cohort of 2009, were given two additional doses of recombinant HBV vaccine between the tenth and the twelfth month of age. A parallel evaluation was conducted on the response to anti-rubella and anti-tetanus vaccine. Only two infants remained non-responders, whereas 68% of the non-responders and 94% of the low responders after the primary vaccination schedule developed antibody titres over 100 mIU ml-1. No significant relationship between the specific antibody level against HBV and against rubella or tetanus 1 month after vaccination was observed.

Human amniotic fluid cells are able to produce IL-6 and IL-8

Problem:  In order to investigate the role of amniotic fluid cells (AFC) in the establishment of feto–maternal immune relationship, we evaluated their phenotype and capacity to produce cytokines.

Neutrophil chemotaxis in infants delivered by caesarean section.

We evaluated polymorphonuclear leucocyte (PMN) chemotaxis and cortisol levels in cord blood from 15 healthy term infants delivered by caesarean section and from 15 healthy vaginally delivered term infants. Mean neutrophil chemotaxis was significantly higher in infants delivered by caesarean section (78.3±23.4μm) than in vaginally delivered infants (57.8±16.6 μm;P=0.01). Mean blood cortisol level was significantly lower in infants delivered by caesarean section (9.14±2.76 μg/dl) than in infants born by vaginal delivery (20.71±6.98 μg/dl;P=0.0001). No relationship was found between PMN chemotaxis and blood cortisol level. The higher neutrophil chemotactic activity observed in infants delivered by caesarean section could be related to general maternal anaesthesia.

B lymphocyte subsets and their functional activity in the early months of life

In the present study we evaluated B-cell subsets and their functional development in 74 newborns from birth to 6 months of life. Moreover, we evaluated “natural antibody” production in vitro. The results documented a predominance of naive B-lymphocytes at all time-points evaluated, decreasing from birth to 6 months (p=0.009). The percentages of CD27+IgD+ and CD27+IgDneg memory B-cells were very low at birth and significantly increased only at 6 months (p=0.02 and p<0.001, respectively). We found a significant increase only in in vitro stimulated IgG production at 6 months as compared to birth (p<0.001). Moreover, a lower secretion of anti-Pn IgM antibodies up to 6 months of age, as compared to controls was observed. Our results underline that the susceptibility and severe course of infection in the neonate can be attributed, at least in part, to the lack of pre-existing immunological memory and competent adaptive immunity.

Antigen-specific T cell response in infants after recombinant hepatitis B virus vaccination at birth: evaluation of T helper lymphocyte diversity

Recombinant hepatitis B virus antigen (rHBsAg)-specific CD4+ T cell clones (TCC) were isolated and expanded from the peripheral blood of nine children vaccinated at birth against the hepatitis B (HB) virus. Four of them responded with protective antibody production (responders), three subjects were unable to produce detectable antibody levels even after revaccination (nonresponders), and two infants produced antibodies only after revaccination (slow responders). TCC were then characterized for their ability to produce cytokines known to be important for T cell expansion (interleukin-2, IL-2) and/or effector functions (IL-4, IFN-gamma, IL-10). Results demonstrated that the frequency of rHBsAg-specific TCC in the samples of nonresponders was comparable to or higher than that in the samples of responders. Nevertheless, the majority of TCC obtained from responders or from slow responders before revaccination displayed the T helper 1 (T(H1))-dominant phenotype, while the majority of TCC obtained from nonresponders were nonpolarized T lymphocytes. After revaccination, the distribution of the different T(H) subsets in slow responders was heterogeneous. Overall, our present data suggest that an absence or delay in developing an rHBsAg-specific antibody response to vaccination is not associated with the capacity to generate an Ag-specific T cell response. However, compared to responders, nonresponding infants react to the rHBsAg vaccination with a reduced capacity to expand and differentiate toward polarized T(H) cells.

Anti-HBV neonatal immunization with recombinant vaccine. Part I. Critical appraisal for a long-lived antibody course.

This study involved 912 infants born to HBsAg-negative mothers from 1 May 1991 to 30 June 1992. The subjects were randomly allocated to an accelerated (Group A) or traditional (Group B) immunization schedule and immunized with 10 micrograms of recombinant HBV vaccine. At the end of the vaccinal cycle 98.14% of both groups were protected against HBV with a high percentage of high responders (88.1% group B and 68% group A). Following a random plan, 345 of the initial 912 infants (144 group A and 201 group B) were serologically evaluated, 15-18 months after the booster dose, to identify the level of long-lasting specific antibody. The data obtained allowed us to identify the non-responder subjects after the seroconversion, to propose the evaluation of antibody titre after the booster dose of vaccine and, because one year after the booster dose 5.6% of the subjects responsive at seroconversion have shown undetectable anti-HBsAg titre, to propose the elevation of the antibody level considered as protective at the end of the vaccinal cycle.