The immune response to diphtheria and tetanus toxoid components of a combined diphtheria tetanus whole-cell pertussis/enhanced inactivated poliovirus (DTwP/eIPV) vaccine, administered in a three-dose schedule to infants at 2, 3\({\raise0.7ex\hbox{$1$} \!\mathord{\left/ {\vphantom {1 2}}\right.\kern-\nulldelimiterspace}\!\lower0.7ex\hbox{$2$}}\) and 10 months of age and followed by a booster at the age of 8 years, was compared with the immune profile of a group of children at the same ages given the customary DTwP vaccine schedule at 2, 4, 6, and 12 months of age and a booster at the age of 8. Diphtheria- and tetanus-antitoxin titers were measured in parallel enzyme-linked immunosorbent assay (ELISA) and radioimmunoassay (RIA). After the reinforcing dose given at 10 months of age, diphtheria antitoxin concentrations of ≥0.01 IU/ml were found in 100% of infants in the study group, 91.7% of whom reached a titer of ≥0.1 IU/ml and a geometric mean titer (GMT) of 0.40 and 0.93 IU/ml in ELISA and RIA, respectively. At 3 and 6 years of age, diphtheria antitoxin values of ≥0.01 IU/ml were detected in 100 and 94% of children with GMT of 0.043 and 0.024 IU/ml, respectively. Seropositivity and GMT values indicative of protection were measured by both ELISA and RIA after the booster at the age of 8 years. Similar results were found in the control group, although the GMT tended to be higher. A good correlation between results obtained by ELISA vs. RIA was evident throughout. Priming at 2 and 3\({\raise0.7ex\hbox{$1$} \!\mathord{\left/ {\vphantom {1 2}}\right.\kern-\nulldelimiterspace}\!\lower0.7ex\hbox{$2$}}\) months with diphtheria and tetanus antitoxin, as a component of a DTwP program, and reinforcing 6 months later induced an immune response indicative of protection against the diseases, which persisted up to the age of the booster recommended at school entry.

We measured total IgG 1, IgG2, IgG3, and IgG 4 concentrations by ELISA in serum (S), total saliva (TS), cervicovaginal secretions (CVS), seminal secretions (SPE), and rectal secretions (RS) from either CDC II/III HIV1-infected subjects or healthy volunteers. Human serum albumin was measured in parallel to calculate the relative coefficient of excretion (RCE). Levels of IgG 1 and IgG 3 directed against gp120 MN also were measured by ELISA in all samples, and the specific activity (SA) calculated. HIV-1-specific IgG2 and IgG 4 were not compared, as total IgG 2 and total IgG4 levels in HIV-1-infected subjects were found to be lower than in the healthy controls. Despite substantial interindividual variability, total IgG 1 and IgG3 concentrations in all fluids were greater in the HIV-1-infected subjects than in the healthy controls. Calculations of RCE indicated predominantly a transudative origin for IgG subclasses in the different mucosal fluids, except for CVS, in which IgG1, IgG2, and IgG4 was produced locally. The transduction behavior of IgG3 in secretions appears to be different from that of other IgG subclasses. HIV-1-infected subjects were considered positive for IgG 1 and IgG3 antibodies against gp120 MN if their antibody levels exceeded the maximum titer measured in the control group. Positive levels of anti-gp120 MN IgG 1 were detected for 100% of HIV-1-infected individuals in S, CVS, and SPE, 97% in TS, and 75% in RS. Fewer subjects had positive levels of IgG3 to gp120 MN in their secretions (maximum 67% in CVS). Despite the low concentrations of total IgG 3, mean SA values for IgG 3 to gp120 M N were greater in secretions than in serum. No significant difference in the SA values for IgG 1 to gp120 MN was observed between the different fluids. Only CVS had a local production of HIV-specific IgG1 Our results highlight the importance of an HIV-specific IgG1 and IgG3 immune response in mucosal fluids from HIV-1-infected subjects.

The ESEN (European Sero-Epidemiology Network) project was established to harmonize the seroepidemiology of five vaccine preventable infections including measles, mumps and rubella in eight European countries. This involved achieving comparability both in the assay results from testing in different centres and also sampling methodology. Standardization of enzyme immunoassay results was achieved through the development of common panels of sera by designated reference centres. The panels were tested at the reference laboratory and then distributed to each participating laboratory for testing using their routine methods. Standardization equations were calculated by regressing the quantitative results against those of the reference laboratory. Our study found large differences in unitage between participants, despite all using an EIA method standardized against an international or local standard. Moreover, our methodology adjusted for this difference. These standardization equations will be used to convert the results of main serosurvey testing into the reference country unitage to ensure inter-country comparability.

Preclinical immunogenicity studies were conducted in rhesus monkeys to determine whether there is immune interference in the response to one or more components of a hexavalent vaccine (Hexavac™) that contains antigens from Haemophilus influenzae (Hib), hepatitis B (HB), diphtheria (D), tetanus (T), acellular pertussis (aP) and inactivated polio virus (IPV). Antibody responses were measured following co-administration of the components at three separate anatomical sites or administration as a hexavalent combination in a single site. After three injections of the hexavalent vaccine, the peak antibody responses to each component of the vaccine were >100-fold above pre-immune titers and persisted at levels >10-fold above pre-immune titers at ≈1 year. Immune interference was observed in the peak response to HB, D and pertussis toxin, but was not seen at later time points. The results indicate that the rhesus monkey model may be useful for pre-clinical evaluation of combination vaccines.

In a single-center double-blind, randomized trial in West Africa, we evaluated the safety and immunogenicity of a new pasteurized, pepsin-digested equine tetanus immunoglobulin (heat-treated equine tetanus immunoglobulin [HT-ETIG]) in the post-exposure prophylaxis of tetanus compared with the reference product, equine tetanus immunoglobulin (ETIG). A total of 134 adults presenting to Garoua Hospital, Cameroon with a tetanus-prone wound were randomized to receive a 3,000 international units (IU) intramuscular injection (deltoid) of either HT-ETIG or ETIG, simultaneously with a tetanus-diphtheria vaccine. No serious adverse reactions were reported. The incidences of local and systemic reactions were similar in the two groups. Repeated measures of equine tetanus-antibody levels measured from Day 0 to Day 28 showed that titers were significantly higher in the HT-ETIG group (P = 0.017). At Day 7, a higher percentage of subjects in the HT-ETIG group had equine antibody levels > or = 0.1 IU/ml (80.4% versus 37.9%; P < 0.0001). No cases of tetanus occurred during the follow-up, attesting to the efficacy of the combined prophylactic treatment.

Enzyme-linked immunosorbent assays (ELISA) were developed to test, in serum and mucosal samples, total IgG, total IgA, serum albumin, and anti-gp120 MN and anti-p24 LAI IgG and IgA levels. These ELISAs were optimized according to reagents and experimental conditions. Inter- and intra-assay coefficients of variation ranged from 3.3% to 18.6%. The ELISA results were linear and precise, and for anti-HIV-1 IgG and IgA, the analytical recovery was close to 100%. For IgG and IgA titration against gp120 MN and p24 LAI, standards were made using pooled sera or gammaglobulins with assigned titres in ELISA units per ml (EU/ml). These standards were used to obtain a linear regression curve that could then be used to obtain the titres of experimental samples. The cut-offs for positivity were determined for sera and mucosal fluid using healthy controls. Validation conditions were defined for ELISAs, and samples that did not satisfy these conditions were retested. Measurement of total IgG and IgA allowed normalization and comparison of the results of specific immunoglobulin levels between different samples. Serum albumin was tested as a marker of transudation from serum to mucosal fluid, allowing calculation of the relative coefficient of excretion, which is one element required to determine the origin of the immunoglobulin detected in mucosal samples. These ELISAs were developed with samples from HIV-1-infected and healthy subjects. We now have the tools to study and understand mucosal immunity in seronegative subjects vaccinated with an HIV-1 candidate vaccine.

We compared IgG and IgA distribution in serum, three different salivary samples, two different rectal secretion samples, cervicovaginal secretions, and seminal secretions from asymptomatic CDC stage II/III HIV-1-infected subjects (n = 44) and from HIV-1-seronegative volunteers (n = 52). In-house ELISAs were used to measure total IgG and total IgA levels, as well as HIV-specific anti-gp120 MN and anti-p24 LAI IgG and IgA. Human serum albumin was titrated in parallel to calculate the relative coefficient of excretion (RCE). In spite of substantial interindividual variability, total IgG concentrations in all fluids were found to be significantly greater in the HIV-1-infected group than in the seronegative subjects. Calculation of RCE values revealed three different types of mucosal secretion: secretions with no local Ig production, such as sperm; secretions with local production of IgA and transudative origin of IgG, such as salivary and rectal samples; and secretions with local production of both IgG and IgA, such as in cervicovaginal secretions. For all mucosal specimens from HIV-1-infected subjects, the response to HIV-1 was predominantly IgG, with highest titers observed in cervicovaginal secretions (although these were lower than serum levels). In contrast, the specific IgA response appeared weaker in the mucosa than in serum.

Between 10 and 11years after children were vaccinated with Vi capsular polysaccharide of Salmonella typhi or meningococcal A+C control vaccine in a double blind randomized trial, we traced 83 subjects, aged 16–20years. A blood sample was taken for determination of Vi antibody titres in both groups by radioimmunoassay. TO and TH titres were also done to assess if the participants had had recent exposure to typhoid fever. Fifty-eight percent of subjects in both groups had protective levels of Vi antibody against Salmonella typhi (a titre greater than 1μgml−1). There was no significant difference in the levels of Vi antibodies in the cases versus the controls (p=0.5). Two of the children who had received meningococcal A+C vaccine had recently had typhoid fever. Our data show that adolescents in typhoid endemic areas have high levels of Vi antibodies regardless of previous vaccination status, suggesting that Vi antibodies are acquired in adolescence by a large percentage of the population in this area. Moreover, Vi vaccination has led to ongoing antibody production in greater than 50% of Vi vaccinated children in an endemic area for a period of 10years. Ongoing antigenic exposure may have contributed to these antibody levels.

As part of the clinical validation process of a new working seed of a licensed yellow fever vaccine (new working seed PV26, Stamaril; Pasteur Mérieux Connaught, Lyon, France), the immunogenicity and safety of two batches of this vaccine (PM-YF) were compared with those of another commercially available vaccine (Arilvax; Evans Medical-Wellcome, Liverpool, United Kingdom) in 211 healthy adults. While the geometric mean titer values at days 10-14 and day 28 after vaccination were higher in the PM-YF group, the vaccines provided equivalent seroprotection (titers > or = 1/10) one month after a single vaccine dose (100% PM-YF versus 99% W-YF; P = 0.001, by one-sided equivalence test). Both vaccines were safe. There were no serious local or systemic reactions reported, nor any clinically significant hepatic function abnormalities associated with the use of either vaccine. These two 17D yellow fever vaccines from different European vaccine manufacturers were highly immunogenic and safe, and provided equivalent seroprotection.

A live recombinant canarypox vector expressing HIV-1 gp120 MN tm/gag/protease LAI (ALVAC-HIV, vCP205) alone or boosted by a p24E-V3 MN synthetic peptide (CLTB-36) was tested in healthy volunteers at low risk for HIV infection for their safety and immunogenicity, Both antigens were well tolerated. ALVAC-HIV (vCP205) induced low levels of neutralizing antibodies against HIV-1 MN in 33% of the volunteers. None of them had detectable neutralizing antibodies against a nonsyncytium-inducing HIV-1 clade B primary isolate (Bx08). After the fourth injection of vCP205, CTL activity was detected in 33% of the volunteers and was directed against Env, Gag, and Pol, This activity was mediated by both CD4(+) and CD8(+) lymphocytes, On the other hand, the CLTB-36 peptide was poorly immunogenic and induced no neutralizing antibodies or CTLs. Although the ALVAC-HIV (vCP205) and CLTB-36 prime-boost regimen was not optimal, further studies with ALVAC-HIV (vCP205) are warranted because of its clear induction of a cellular immune response and utility as a priming agent for other subunit antigens such as envelope glycoproteins, pseudoparticles, or new peptides.

Combined pediatric vaccines have the advantages of conferring protection against multiple common infectious diseases with a reduced number of injections. Their use should lead to better compliance to recommended vaccination schedules. Diphtheria (D), tetanus (T) and whole-cell pertussis vaccine (P) have been successfully combined, with or without inactivated poliovirus vaccine (IPV) in the same syringe for many years. Recently developed acellular pertussis (aP) Haemophilus influenzae type B (Hib), inactivated poliomyelitis virus and hepatitis B vaccines are ideal candidates for inclusion in current combined vaccines. Nevertheless, the development of new combinations has to face preclinical and clinical issues: the appropriate formulation of the new antigen(s) and other vaccine components needs to be determined to ensure compatibility and guard against potential additive or unexpected adverse reactions; potential immunological interference between antigens and the negative impact of other vaccine components on immunogenicity may occur, and these have to be examined also. Whole-cell pertussis vaccines are highly protective against whooping cough, but the severe adverse reactions that these vaccines sometimes produce have led to hesitation over their use, including the decision of some countries to stop pertussis immunization. To increase the acceptability of pertussis vaccination, Pasteur Mérieux Connaught has developed a combined D, T and a two-component acellular pertussis vaccine (DTaP), composed of purified pertussis toxoid (PT) and filamentous haemagglutinin (FHA), which has been shown to be effective in an efficacy trial conducted in Senegal. Acellular DTaP vaccines are immunogenic and have a better safety profile than DTP vaccines, when given either for the primary series, for the booster vaccination or for both. In order to meet worldwide demands, the combined DTaP–IPV or DTP–IPV has been developed for countries where IPV is recommended. Following the encouragement of the WHO, an H. influenzae type B tetanus-conjugated (Act–HIB) vaccine, has been combined in a full liquid formulation with the whole-cell DTP. This vaccine showed a good safety and immunogenicity profile in infants and in toddlers. A combined DTaP–IPV–PRP–T vaccine (where the Act-HIB vaccine is reconstituted by the full-liquid DTaP–IPV) also has been successfully developed both for the primary series and for booster vaccination; although, a reduced immunogenicity against PRP observed after the primary series, this did not affect vaccine priming. Hepatitis B immunization campaigns targeting high-risk groups have failed to control the disease in areas of low endemicity. In 1992, the WHO recommended that hepatitis B vaccination should be integrated into the EPI in all countries by 1997–1999. For that purpose, hepatitis B vaccine is currently evaluated in pediatric combined vaccines. Developing new combination vaccines is a difficult but essential process for maintaining high immunization rates worldwide against infectious diseases, provided that the costs are acceptable. New combined vaccines including pneumococcal and meningococcal component are under wide-scale development.

BACKGROUND:In recent years additional parenteral vaccines have been recommended for routine immunization of infants in the US and elsewhere. The ability to administer multiple vaccines as a single injection without unacceptably increasing reactogenicity or decreasing immunogenicity of any component would offer many practical advantages.METHODS:A randomized, open, controlled trial was conducted to assess the tolerance profile and immunogenicity, as well as to identify potential antigenic interferences, resulting from administration of a parenteral combination vaccine for infants. The vaccine contains diphtheria and tetanus toxoids, acellular pertussis antigens (DTaP), enhanced inactivated poliovirus (eIPV) and Haemophilus influenzae type b-tetanus toxoid conjugate (PRP-T). Infants (n=711) were randomly assigned to receive 1 of 5 regimens as the primary series at 2, 4 and 6 months of age, by group: (1) DTaP plus oral polio vaccine (OPV); (2) DTaP plus eIPV (separate injections); (3) DTaP-eIPV combined as a single injection; (4) DTaP-eIPV combined, plus a separate injection of PRP-T; or (5) DTaP-eIPV combined and reconstituting PRP-T, as a single injection. At 3, 5 and 7 months Groups 1, 2 and 3 received PRP-T. At 12 months all infants received a booster dose of DTaP reconstituting PRP-T as a single injection, plus a separate injection of measles, mumps and rubella vaccine. Groups 2, 3, 4 and 5 received OPV at 7 months, and all infants received OPV at 13 months. Serum immune responses were measured to the primary series at 2 and 7 months and to the booster dose at 12 and 13 months.RESULTS:Reaction rates were similar among groups. In the primary series combining eIPV with DTaP decreased geometric mean titers (GMTs) to diphtheria, tetanus and pertussis. In addition concomitant PRP-T (either simultaneous or combined) with DTaP-eIPV lowered anti-PRP and further decreased tetanus GMTs. Nonetheless in 100% of infants protective titers were achieved against diphtheria and tetanus (>0.01 IU/ml each) and against the poliovirus types 1, 2 and 3 after eIPV (Groups 2 to 5); 99% of infants (Groups 4 and 5) had protective titers against PRP (> or = 0.15 microg/ml). After boosting with DTaP/PRP-T decreased GMTs to diphtheria and PRP antigens were observed in the groups that received DTaP and eIPV combined. Nonetheless protective titers to diphtheria, tetanus and PRP occurred consistently. In contrast concomitant PRP-T with DTaP-eIPV enhanced the pertussis GMTs.CONCLUSIONS:We conclude that combined DTaP, eIPV and PRP-T in a single injection is well-tolerated and elicits an acceptable immune response to each component.

Subjects (n = 312) received either the human diploid cell rabies vaccine (HDCV) or the purified Vero cell rabies vaccine (PVRV) according to either two-injection (days 0 and 28) or three-injection (days 0, 7, and 28) primary regimens. They received a booster injection at 1 year. Rabies antibody levels were measured after the primary series and the booster and then each year for the next 10 years. The results confirm the superior long-term immunogenicity of the three-injection over the two-injection protocol. HDCV and PVRV in three doses were equally immunogenic. A booster injection at 1 year provides long-term seroconversion (titer > or = 0.5 IU/mL). Antibody titers 2 weeks after the 1-year booster allowed prediction of long-term immunity. Good responders, with titers > or = 30 IU/mL, were protected for at least 10 years. An algorithm for differentiation between good responders and poor responders with respect to vaccine booster strategies is proposed.

METHODS:In an open randomized study we compared the safety and immunogenicity of two schedules for priming and booster vaccinations of infants. A pentavalent combination vaccine, including a lyophilized Haemophilus influenzae type b-tetanus toxoid conjugate vaccine reconstituted with a liquid diphtheria, tetanus, acellular pertussis (pertussis toxoid and filamentous hemagglutinin) and inactivated polio vaccine (DTaP-IPV/Act-HIB; Pasteur Mérieux Connaught, Lyon, France) was administered to 236 Swedish infants either at 2, 4 and 6 months or at 3 and 5 months, and a booster dose was administered 7 months after the last primary dose. Adverse events were monitored by diaries for 3 days after each vaccination and by questions at the ensuing visits. Antibodies against the different vaccine components were analyzed after the primary series of vaccinations, before and after the booster injections. RESULTS:There were no serious adverse reactions, and the rates of febrile events and local reactions were low in both groups. The three dose primary schedule induced higher geometricmean concentrations for all antigens than did the two dose schedule, but there were no differences between the groups in proportions with protective antibody titers against diphtheria, tetanus, Hib and polio or in proportions with certain defined levels of pertussis antibodies. Prebooster results showed a similar pattern, with the exception that the group primed with three injections showed higher proportions of infants with detectable antibodies against polio-virus types 1 and 3. After booster vaccinations there were no differences between the two schedules in geometric mean or in proportions with antibodies above defined antibody concentrations, indicating effective priming from both primary series of vaccinations. Conclusion. The combined vaccine DTaP-IPV/ Act-HIB vaccine was equally safe and immunogenic when administered according to both time schedules studied.

To evaluate the potential protective efficacy of a live recombinant human immunodeficiency virus type 1 (HIV-1) canarypox vaccine candidate, two chimpanzees were immunized five times with ALVAC-HIV-1 vCP250, a recombinant canarypox virus that expresses the HIV-1IIIB(LAI)gp120/TM,gag,and protease gene products. One month after the last booster inoculation, the animals were challenged by intravenous injection of cell-associated virus in the form of peripheral blood mononuclear cells from an HIV-1IIIB(LAI)-infected chimpanzee. One chimpanzee with a neutralizing antibody titer to HIV-1IIIB(LAI)of 128 at the time of challenge was protected, whereas both the second animal, with a neutralizing antibody titer of 32, and a naive control animal became infected. At 5 months after challenge, the protected chimpanzee and a third animal, previously immunized with various HIV-1MNantigens, were given a booster inoculation. The two animals were challenged intravenously 5 weeks later with twenty 50% tissue culture infectious doses of cell-free HIV-1DH12, a heterologous subtype B isolate. Neither chimpanzee had neutralizing antibodies to HIV-1DH12, and neither one was protected from infection with this isolate. The immune responses elicited by vaccination against HIV-1IIIB(LAI)or HIV-1MNdid not, therefore, protect the animals from challenge with the heterologous cell-free HIV-1DH12.

Sera from subjects vaccinated with the Pasteur Merieux (PM) inactivated hepatitis A vaccine (AVAXIM) have been analysed in order: (1) to assess comparability of the results provided by a modified radio-immunoassay (mRIA) in the different laboratories involved in testings of sera during clinical trials and by enzyme-linked immunoabsorbent assay (ELISA) used during development of other inactivated hepatitis A vaccines; (2) to describe the IgM responses elicited by this vaccine compared with one control vaccine [HAVRIX, 720 ELISA antigens units; Smithkline Beecham (SB), Rixensart, Belgium]; (3) to provide comparative data between the PM vaccine and the SB vaccine on neutralizing activities of vaccine-induced antibodies. Vaccine-induced antibody titres evaluated by a mRIA in different laboratories correlated well and validated the comparability of the results obtained in various vaccine trials conducted by PM. Geometric mean titres (GMTs) expressed as milli-international units (mlU/ml) were higher with ELISA especially after the first dose, but seroconversion rates were similar and a good correlation was found between the two assays. IgM vaccine-induced antibodies were detectable in nearly all vaccinated subjects from week 2 after vaccination, with a peak titre between weeks 2 and 4 after the first dose. Comparison of GMTs by the Student Fisher t-test was statistically significant (P < 0.05) only at week 2 with higher titres in PM vaccinees. Neutralizing antibodies were detected after vaccination with the PM inactivated hepatitis A vaccine. The titres gradually increased between the second week after the first dose and the booster dose (week 24). A strong booster effect of the second dose on neutralizing titres was observed. Seroneutralizing titres induced at week 2 in subjects vaccinated with the PM inactivated hepatitis A vaccine were statistically significantly higher (P < 0.05) from those induced by the SB vaccine.

Depuis 1988, un programme de prevention et de vaccination contre l’hepatite B, utilisant un vaccin recombinant produit sur cellules de mammiferes (Genhevac B®) a ete mis en place au benefice des nouveau-nes de l’archipel des Australes, zone pilote de la lutte contre l’hepatite B en Polynesie francaise. Trois calendriers vaccinaux ont ete utilises : l’un a quatre doses aux mois M0 (jour de naissance), M1, M2, M12 et les deux autres a trois doses M0, M1, M6 ou M0, M1, M12. L’evaluation serologique de ce programme a ete effectuee annuellement durant 5 ans. Parmi les 837 enfants ayant recu au moins une dose de vaccin, 5 sont porteurs de l’antigene HBs. Apres une ou deux doses de vaccin, respectivement 88 et 97 % des enfants avaient des taux seroprotecteurs d’anticorps anti-HBs et/ou d’anticorps anti-PreS2. Apres trois doses de vaccin, les taux de seroprotection et les moyennes geometriques des anticorps anti-HBs etaient respectivement de 95% et de 217 mUI/ml avec le calendrier M0, M1, M2 ; 92 % et 389 mUI/ml avec le calendrier M0, M1, M6 et 93 % et 344 mUI/ml avec le calendrier M0, M1, M12. Apres quatre doses de vaccin (M0, M1, M2, M12), les donnees correspondantes sont de 100 % et 1 228 mUI/ml. Parmi les 18 enfants vaccines nes de mere double porteuse AgHBs-AgHBe, un seul etait porteur de l’Ag HBs permettant d’evaluer le taux de protection de la transmission perinatale a 94 %. Ces resultats suggerent que, dans les conditions de terrain, une vaccination systematique des nouveau-nes avec le Genhevac B® sans utilisation d’immunoglobulines specifiques confere une protection efficace, precoce et durable. Le calendrier vaccinal recommande en Polynesie francaise est M0, M1, M6-M12 (3e dose entre M6 et M12), suivi d’une injection de rappel a l’âge de 6 ans. La vaccination systematique des nouveau-nes a ete generalisee depuis le 1er avril 1992 a tous les archipels de Polynesie francaise. Un programme de rattrapage pour la tranche d’âge 4-10 ans a debute, utilisant le meme calendrier d’immunisation.