Initiation of HAART in drug-naive HIV type 1 patients prevents viral breakthrough for a median period of 35.5 months in 60% of the patients.

The introduction of potent combinations of antiviral drugs is a major breakthrough in the treatment of HIV. We investigated the long-term virologic outcome and the development of resistance after initiating highly active antiretroviral therapy (HAART) in drug-naive patients in daily clinical practice. Twenty-five treatment-naive HIV-1 patients were started on HAART. Fifteen patients responded with a drop in viral load below the limit of detection during 35.5 (interquartile range: 7) months of therapy. In 6 of 10 patients with virologic failure, virus with resistance-related mutations against the received drugs emerged. Compared with responders (R), nonresponding (NR) patients were in a later disease stage at therapy start (p = 0.0089) with lower CD4 cell counts at baseline (p = 0.040), and a lower proportion of nonresponders showed protease inhibitor (PI) levels above C(min) (p = 0.049). More NR patients showed secondary PI mutations at baseline (p = 0.079), and the CCR2-64I coreceptor polymorphism was absent among NR patients, compared with 38.5% of R patients displaying CCR2-64I (p = 0.053), although the differences were not significant. In conclusion, starting HAART in antiretroviral drug-naive HIV-infected patients followed in daily clinical practice prevented viral breakthrough for up to 44 months in 60% of the patients. Virologic failure was associated with the development of resistance-related mutations, a later stage of disease at start of therapy and lower PI drug levels.

Postscript relating to new allegations made by Edward Hooper at The Royal Society Discussion Meeting on 11 September 2000.

At The Royal Society Discussion Meeting, Origins of HIV and the AIDS epidemic, which this issue records, Edward Hooper added two new 'smoking guns' to the accusations published previously in The river. These were proposed as conclusive evidence for the hypothesis that simian immunodeficiency virus-contaminated CHAT polio vaccine caused the HIV-1 group M epidemic. We have investigated the facts in relation to these 'smoking guns'.

The Jezierski papers: live polio vaccine development in colobus monkey cells but not chimpanzee cells in the Belgian Congo, 1952-1958.

A reading of ten relevant papers by Alexandre Jezierski provides evidence for the only attempt in Central Africa to develop a live oral polio vaccine (OPV) from growing reference wild polio strains to 210 passages in colobus monkey tissue culture, and experimental administration to about 25 humans. Chimpanzees were used as a human model, but their tissues or kidneys were absent from the passage and production line of the proposed vaccine. Thus, the implication published by Hooper that Jezierski had produced a candidate OPV that might have contained chimpanzee viruses, possibly simian immunodeficiency virus cpz or the precursor of human immunodeficiency virus-1 group M, is incorrect.

Prevalence of genotypic resistance among antiretroviral drug-naive HIV-1-infected patients in Belgium.

OBJECTIVES: To estimate the prevalence and the evolution over time (1995-1998) of genotypic resistance to antiviral drugs in antiretroviral drug-naive HIV-1-infected patients in Belgium. DESIGN: Belgian Aids Reference Laboratories provided retrospective samples and clinical data from antiretroviral drug-naive HIV-1-infected patients who visited the hospital for the first time in 1995 (n=45), 1997 (n=75) and 1998 (n=111). Genotypic resistance to the three available classes of drugs was monitored using the Line Probe Assay (Innogenetics, Gent, Belgium). Additionally, ARMS-151 was performed for scoring multinucleoside resistance. RESULTS: The prevalence of genotypic resistance at baseline to nucleoside analogue reverse transcriptase inhibitors (NRTIs) and non-nucleoside reverse transcriptase inhibitors (NNRTIs) were each between 10% and 20% for 1995, 1997 and 1998 without an increasing trend over time. For NRTIs, resistance mutations were mainly related to zidovudine in 1995, whereas in 1997 and 1998 baseline resistance was scored for zidovudine, lamivudine or for both drugs simultaneously. No patients displayed the multi-nucleoside resistance Q151M mutation. Baseline resistance mutations to protease inhibitors (PIs) did not rise significantly: 4.4% in 1995, 8% in 1997 and 9.9% in 1998. When scoring any resistance-related mutation, 26.6% displayed genotypic baseline resistance in 1995, 26.6% in 1997 and 31.5% in 1998. DISCUSSION: The prevalence of genotypic baseline resistance to any drug, as scored with LiPA, in naive HIV-1 patients in Belgium is 29%, with baseline resistance mutations to one or several drugs from all available classes of antiviral drugs. The ability of LiPA to pick up minor variants could be an explanation for the higher overall prevalence we observe, when compared to recent estimates in other countries of 16.3% and 22%, which were based on sequencing methods. According to the European guidelines for resistance testing, resistance testing in Belgium before starting antiviral therapy should be considered.

Mutations in the non-nucleoside binding-pocket interfere with the multi-nucleoside resistance phenotype.

OBJECTIVES: To investigate the genotypic and phenotypic effects of in vitro resistance selection with lamivudine and/or the second generation non-nucleoside reverse transcriptase inhibitor (NNRTI) quinoxaline HBY097 using HIV-1 isolates carrying the multi-nucleoside resistance pattern linked to the Q151M mutation. METHODS: Virus strains were selected in C8166 cells in the presence of increasing concentrations of lamivudine or HBY097. In parallel control experiments, the virus was cultured in C8166 cells in the absence of drugs. The entire reverse transcriptase encoding region was amplified using polymerase chain reaction and was subsequently sequenced. Antiviral activities of drugs were evaluated in C8166 cells. RESULTS: High-level resistant viruses were selected rapidly in the presence of lamivudine and quinoxaline (less than 10 passages). The multi-nucleoside resistance mutations were stable during in vitro resistance selection. Lamivudine elicited the acquisition of the M184I mutation. Phenotypic resistance to all nucleoside-analog reverse transcriptase inhibitors (NRTIs) was increased when M184I was added to the multi-nucleoside resistance background in the absence of NNRTI-resistance mutations. In most cases of HBY097 resistance selection, at least two mutations associated with NNRTI resistance resulted in high-level NNRTI resistance. The NNRTI resistance-related mutations partially reversed the phenotypic resistance to most NRTIs, except to abacavir. The addition of the M184I mutation to the NNRTI-multi-nucleoside resistance set abolished this antagonizing effect for didanosine, zalcitabine and lamivudine, but further potentiated the phenotypic reversal for zidovudine and stavudine. CONCLUSION: Changes in the non-nucleoside binding pocket must affect the conformation of residues at the dNTP binding site, and can result in a partial phenotypic reversal of the multi-nucleoside resistance phenotype.

In vitro evaluation of the effect of temporary removal of HIV drug pressure.

We tried to establish whether MT-4 cells that were infected with HIV-1(HTLV-III(B)) at a high multiplicity of infection (m.o.i.=1), and subsequently treated with high concentrations of anti-HIV drugs for several days, would be able to resume virus production after the antivirals are washed away. The HIV inhibitors studied were the nucleoside reverse transcriptase inhibitors (NRTIs) zidovudine and lamivudine, the non-nucleoside reverse transcriptase inhibitors (NNRTIs) nevirapine, delavirdine and loviride, the acyclic nucleoside phosphonate RT inhibitor (R)-9-(2-phosphonylmethoxypropyl)adenine (tenofovir) and the protease inhibitors (PIs) saquinavir, indinavir and ritonavir. The compounds, at 50 and 500 times their 50% inhibitory concentration (IC(50), determined at a m.o.i.=0.01), were added immediately after virus adsorption and removed after an incubation period of 0 (wash control), 24, 48 or 72 h. Virus breakthrough was monitored by microscopical examination of cytopathicity, viral infectivity (yield) and p24 levels in the supernatant. The presence of HIV-1(HTLV-III(B)) proviral DNA was determined after a 72-h incubation period. None of the antiviral drugs studied was able to prevent resumption of viral growth after removal of the compound. Tenofovir, lamivudine and the NNRTIs nevirapine, delavirdine and loviride, at 500 times their respective IC(50), were able to delay viral breakthrough for approximately 2-3 days. The NRTI zidovudine and the PIs saquinavir, indinavir and ritonavir, under the same conditions, were not able to delay viral breakthrough at all. Virus recovered upon treatment proved as sensitive to the anti-HIV drugs as wild-type virus. Our results suggest that viral replication at the cellular level was not completely inhibited by drug monotherapy. Consequently, virus rebounded when drug therapy stopped. In conclusion, our findings suggest that drug holidays would result in viral breakthrough, even after virus replication has been previously suppressed by adequate drug levels.

Presence of 2',5'-Bis-O-(tert-butyldimethylsilyl)-3'-spiro-5"-(4"-amino-1",2"-oxath iole-2",2"-dioxide) (TSAO)-resistant virus strains in TSAO-inexperienced HIV patients.

HIV-1 samples from six patients undergoing diverse anti-HIV therapies possessed the E138A mutation in their reverse transcriptase (RT) genome. Patients were receiving the following therapies: TIBO monotherapy (one patient); zidovudine plus didanosine combination therapy (one); zidovudine monotherapy (one); sequential therapy with zidovudine, then stavudine and finally zalcitabine plus didanosine (one); and two were drug naive. E138K, not E138A, is a known TSAO-specific resistance mutation, emerging under selective pressure in vitro. Our phenotypic data on the patient isolates, confirmed by data on an E138A mutant acquired through in vitro mutagenesis, indicated that an alanine substitution for glutamate at codon 138 of the HIV-1 RT renders the virus TSAO resistant, confirming the importance of this amino acid residue in the activity of TSAO derivatives. In addition, we have demonstrated through phenotypic analysis of the E138A and A98S mutants (after in vitro mutagenesis) that the mutation A98S, found in one of these patients, could be partially responsible for the phenotypic reversal of TSAO resistance. This reversal could be explained by the restoration of a hydrogen bond between 98S and the main-chain residue L349, which compensates for the loss of the E138-G99 main-chain hydrogen bond. As TSAO derivatives have not been used in the clinical setting, the presence of the E138A mutation at a frequency of 6.7% in our study of 90 TSAO-inexperienced HIV-seropositive individuals implies that 138A of the RT must be a natural variant and that the mutant virus is replication competent. Our observations suggest that the E138A mutation may likely arise in patients under the selective pressure of TSAO or related compounds that show a decreased antiviral potency toward the E138A variant.

Baseline HIV type 1 genotypic resistance to a newly added nucleoside analog is predictive of virologic failure of the new therapy.

We evaluated the predictive value of baseline HIV-1 genotypic resistance mutations for failure of a nucleoside reverse transcriptase inhibitor (NRTI) containing therapy. The change in therapy of 88 HIV-1-infected patients was analyzed retrospectively, relating the genotypic resistance profile at baseline to the evolution of viral load and CD4+ T cell counts. Genotypic resistance at baseline and at 6 months was evaluated with the LiPA HIV-1 RT, which detects mutations at codons 41, 69, 70, 74, 184, and 215. At 1 to 3 months after change in therapy, patients without preexisting resistance mutations to the new drug (group S) had a significantly better evolution in viral load (reduction of 0.37 log(10)) compared with patients with known preexisting resistance mutation(s) (group R) (increase of 0.08 log(10)). This difference was particularly striking for patients with the baseline M184V mutation and whose treatment was modified by the addition of lamivudine. After 6 months the median difference in viral load evolution between the two groups increased to 0.61 log(10): the viral load of patients of group S was still 0.18 log(10) below baseline while patients of group R had an increase of 0.43 log(10) in viral load above baseline. Changes in CD4+ T cell counts were not significantly different. The evolution in viral load in HIV-1-infected patients with and without baseline resistance mutation(s) toward a newly added NRTI is significantly different at 1-3 months and at 6 months after changing or adding one NRTI.

Tempo and mode of human and simian T-lymphotropic virus (HTLV/STLV) evolution revealed by analyses of full-genome sequences.

We investigated the tempo and mode of evolution of the primate T-lymphotropic viruses (PTLVs). Several different models of nucleotide substitution were tested on a general phylogenetic tree obtained using the 20 full-genome HTLV/STLV sequences available. The likelihood ratio test showed that the Tamura and Nei model with discrete gamma-distributed rates among sites is the best-fitting substitution model. The heterogeneity of nucleotide substitution rates along the PTLV genome was further investigated for different genes and at different codon positions (cdp's). Tests of rate constancy showed that different PTLV lineages evolve at different rates when first and second cdp's are considered, but the molecular-clock hypothesis holds for some PTLV lineages when the third cdp is used. Negative selection was evident throughout the genome. However, in the gp46 region, a small fragment subjected to positive selection was identified using a Monte Carlo simulation based on a likelihood method. Employing correlations of the virus divergence times with anthropologically documented migrations of their host, a possible timescale was estimated for each important node of the PTLV tree. The obtained results on these slow-evolving viruses could be used to fill gaps in the historical records of some of the host species. In particular, the HTLV-I/STLV-I history might suggest a simian migration from Asia to Africa not much earlier than 19,500-60,000 years ago.

High prevalence of GB virus C/hepatitis G virus in Kinshasa, Democratic Republic of Congo: a phylogenetic analysis.

A prevalence of 10.3% of GB virus C (GBV-C)/hepatitis G virus (HGV) carriers was found in 97 pregnant women from Kinshasa, Congo (formerly Zaire), while prevalences of 1%, 4.1%, and 0% were found for hepatitis C virus, human immunodeficiency virus, and human T-lymphotropic virus respectively. Phylogenetic analysis of the ten GBV-C/HGV positives based on the 5' non-coding region using three different methods identified consistently three GBV-C/HGV genotypes. Four main clades were found within the type 1 sequences. All the Congolese isolates are GBV-C/HGV type 1 in two different clades. The clustering of seven Congolese isolates was inconsistent in different methods. Further likelihood-mapping analysis showed a well-resolved phylogeny, confirming the clustering of the seven Congolese isolates with a Belgian strain representing a new clade in the GBV-C/HGV type 1 sequences.

Evaluating Clinical Isolates for Their Phenotypic and Genotypic Resistance Against Anti-HIV Drugs.

The high replication rate of HIV, together with the low fidelity of its reverse transcriptase, provides the virus with an unprecedented genomic flexibility. This allows a fast adaptation to selective pressure, including antiviral drugs, resulting in the development of drug-resistant strains. The present improvements in the treatment of AIDS patients are at least partly owing to antiviral therapy. To assess the implications of HIV drug resistance on patient management, drug resistance assays for clinical HIV isolates are widely being used. Ideally, monitoring drug resistance should help clinicians in their treatment decisions. If patients would really benefit clinically from this strategy, then the gain from clinical improvement and from omitting drugs to which the virus is already resistant would outweigh the cost of drug resistance testing. In the next few years, researchers should consolidate the clinical benefit of antiviral drug resistance testing, and for this they need fast, reliable and cheap assays. All present assays are in vitro assays, which can only partly mimic the in vivo situation with confounding factors, such as cellular resistance (1). Efforts are presently made to establish in vivo assays (2; see also Chapter 10 ).

Resistance of the human immunodeficiency virus to the inhibitory action of negatively charged albumins on virus binding to CD4.

Negatively charged albumins (NCAs) have been identified as potent inhibitors of HIV-1 replication in vitro. Time of addition studies suggest that succinylated and aconitylated human serum albumin (Suc-HSA and Aco-HSA) act at an early stage of the virus life cycle, and surface plasmon resonance (BIAcore) experiments have confirmed a direct interaction of NCAs with HIV-1 gp120. Resistance to Suc-HSA and Aco-HSA was analyzed by characterizing HIV-1 variants that were selected in cell culture after serial passage of the NL4-3 strain in the presence of the compounds. After 24 passages (126 days) we isolated variants that were resistant to Suc-HSA (>27-fold) and Aco-HSA (37-fold), as compared with the wild-type NL4-3 virus. The binding of the NCA-resistant HIV strains to CD4+ MT-4 cells could no longer be inhibited by either Suc- or Aco-HSA. The emergence of mutations in the envelope gp120 of the resistant virus paralleled the emergence of the resistant phenotype. The Suc-HSA-resistant strain was 100-fold cross-resistant to the G quartet-containing oligonucleotide AR177 (Zintevir, an HIV-binding inhibitor), and partially cross-resistant to dextran sulfate, but remained sensitive to the bicyclam AMD3100 and the chemokine SDF-1alpha, which block HIV replication by interaction with the chemokine receptor CXCR4. Furthermore, neither Suc-HSA nor Aco-HSA inhibited the binding of monoclonal antibodies 12G5 and 2D7 (directed to CXCR4 and CCR5, respectively) in SUPT-1 cells or THP-1 cells. These results confirm that NCAs bind primarily to gp120 and do not interact directly with the HIV chemokine receptor but block the binding of the virus particles (through gp120) with CD4+ cells.

Different population dynamics of human T cell lymphotropic virus type II in intravenous drug users compared with endemically infected tribes.

The phylogeny of human T cell lymphotropic virus type II (HTLV-II) was investigated by using strains isolated from Amerindian and Pygmy tribes, in which the virus is maintained primarily through mother-to-child transmission via breast-feeding, and strains from intravenous drug users (IDUs), in which spread is mainly blood-borne via needle sharing. Molecular clock analysis showed that HTLV-II has two different evolutionary rates with the molecular clock for the virus in IDUs ticking 150-350 times faster than the one in endemically infected tribes: 2.7 x 10(-4) compared with 1.71/7.31 x 10(-7) nucleotide substitutions per site per year in the long terminal repeat region. This dramatic acceleration of the evolutionary rate seems to be related with the mode of transmission. Mathematical models showed the correlation of these two molecular clocks with an endemic spread of HTLV-II in infected tribes compared with the epidemic spread in IDUs. We also noted a sharp increase in the population size of the virus among IDUs during the last decades probably caused by the worldwide increase in intravenous drug use.

The discovery of two new divergent STLVs has implications for the evolution and epidemiology of HTLVs.

We have isolated and characterised two divergent simian T-lymphotropic viruses (STLV), not belonging to the established human and simian T-lymphotropic virus lineages HTLV-1/STLV-1 and HTLV-2. STLV-L, from an Eritrean sacred baboon (Papio hamadryas), has been typed as a third type of simian T-lymphotropic virus, distinct from HTLV-1/STLV-1 and HTLV-2. The other virus, isolated from Congolese bonobos (Pan paniscus), is a distinct member of the HTLV-2 clade and has been designated STLV-2. The isolation of these two simian viruses shows that the spectrum of HTLVs/STLVs is larger than previously expected. Our data indicate that the two lineages STLV-L and HTLV-2/STLV-2 are of African origin, while the HTLV-1/STLV-1 lineage has been shown to be of Asian origin. These data, together with our phylogenetic analyses, suggest an African origin of the HTLV/STLV ancestor, which provides new clues about virus dissemination. Furthermore, the atypical serological profiles exhibited by STLV-L or STLV-2 infected animals in western blot, raise questions about the efficiency of current screening methods to type highly divergent HTLVs/STLVs. Considering the growing interest in xenotransplantations, more epidemiological and biological knowledge of simian and human T-lymphotropic viruses is necessary to estimate the risk of interspecies transmissions.

Activity of non-nucleoside reverse transcriptase inhibitors against HIV-2 and SIV.

BACKGROUND: After the initial discovery of 1-(2-hydroxyethoxymethyl)-6-(phenylthio)thymine (HEPT) and tetrahydroimidazo[4,5,1-jk][1,4]benzodiazepin-2(1H)-one and thione (TIBO) derivatives, several other non-nucleoside reverse transcriptase (RT) inhibitors (NNRTI), including nevirapine (BI-RG-587), pyridinone derivatives (L-696,229 and L-697,661), delavirdine (U-90152), alpha-anilinophenylacetamides (alpha-APA) and various other classes of NNRTI have been described. The hallmark of NNRTI has been based on their ability to interact with a specific site ('pocket') of HIV-1 RT. OBJECTIVE: To investigate whether, in addition to HIV-1, different strains of HIV-2 (ROD and EHO) and SIV (mac251, agm3 and mndGB1) are sensitive to a selection of NNRTI i.e. delavirdine, the HEPT derivative I-EBU (MKC-442), 8-chloro-TIBO (tivirapine), alpha-APA (loviride), nevirapine and the pyridinone derivative L-697,661. METHODS AND RESULTS: The NNRTI tested inhibited the replication of the different strains of HIV-2 and SIV at micromolar concentrations. The inhibitory effects of the NNRTI on HIV-2-induced cytopathicity correlated well with their inhibitory effects on HIV-2 RT activity. Drug-resistant HIV-2 (EHO) variants containing the Ser102Leu and/or Glu219Asp mutations in their RT were selected after passaging the virus in MT-4 cells in the presence of increasing concentrations of delavirdine. The EHO virus mutants were at least 20-fold less susceptible to the antiviral effects of delavirdine. Some cross-resistance, depending on the mutant strain, was observed with the other NNRTI tested (i.e. MKC-442, tivirapine, loviride and pyridinone L-697,661). CONCLUSIONS: Our data demonstrate that NNRTI are not exclusively specific for HIV-1 but are also inhibitory to different HIV-2 and SIV strains. These observations will have important implications for the development of new NNRTI with higher activity against both HIV-1 and HIV-2. Furthermore, in view of their anti-SIV activity, NNRTI could be evaluated further for their in vivo anti-retrovirus efficacy in non-human primate models.

The origin and evolution of human T-cell lymphotropic virus type II (HTLV-II) and the relationship with its replication strategy.

In this review, the origin and evolution of the human T-cell lymphotropic virus type II (HTLV-II) are discussed, with particular emphasis on its high genomic stability. In particular, it appears that the virus originated in the African continent and has been infecting human populations for several thousands of years. The very low divergence accumulated on average between different viral strains during such a long period could be explained by considering that in infected individuals the viral amplification could be due mainly to the clonal expansion of the infected cells, via cellular mitosis, rather than to reverse transcription. HTLV-II was introduced into the American continent during one or more migrations of HTLV-II-infected Asian populations over the Bering land bridge, some 15,000-35,000 years ago. Finally, during the last few decades, HTLV-II has been transmitted from native Amerindians to injecting drug users (IDUs). It might be speculated that at least two separate introductions of HTLV-II in European IDUs from US IDUs have occurred, due to the practice of needle-sharing among IDUs.

The effect of reconstitution of an Haemophilus influenzae type b-tentanus toxoid conjugate (PRP-T) vaccine on the immune responses to a diphtheria-tetanus-whole cell pertussis (DTwP) vaccine: a five-year follow-up.

Controversial results have been obtained from previous studies on the combined administration of Haemophilus influenzae type b-tetanus toxoid conjugate (PRP-T) and diphtheria-tetanus-whole-cell pertussis (DTwP) combination vaccines, with regard to possible reciprocal interference between the constituent antigens. To document the priming effect and possible long-term immunogenic interference of PRP-T and DTwP combination vaccines, a randomized, double-blind, controlled study was conducted in Belgium. A total of 168 healthy infants received, at 3, 4 and 5 months of age, DTwP vaccine mixed just prior to injection either with PRP-T vaccine (group A, DTwP//PRP-T, N = 85) or with placebo (group B, DTwP//Placebo, N = 83). At the age of 14 months, children of both groups were randomized to receive either a dose of DTwP//PRP-T vaccine (subgroups A1 and B1) or a dose of Hib polysaccharide (PRP) vaccine (subgroups A2 and B2). Those children in subgroups A1 and B1 had an additional serum sample taken at the age of 5 years (at the time of a DT booster). The immune response to Hib polysaccharide at the age of 4, 5 and 6 months confirmed the excellent immunogenicity profile of PRP-T in infants. In addition, the vigorous anamnestic response (i.e. a 20-fold increase of GMT) to a booster dose of the plain capsular polysaccharide (PRP) reflected the efficient Hib-priming induced by the combined DTwP//PRP-T vaccine. Reconstitution of PRP-T with DTwP did not affect the immune response to diphtheria toxoid or pertussis agglutinins. Nevertheless, at almost any time point during the five-year follow-up, the tetanus antitoxin GMT values were significantly lower in the DTwP//PRP-T group (A and A1) than in the DTwP//Placebo group (B and B1). Despite the suppressive effect on GMT values, intergroup differences in rates of seroprotection were never significant, except after doses 2 and 3 for which there were lower percentages of children in group A with antitoxin titers > 0.05 IU/mL and > 1.0 IU/mL. In the group primed with the combined DTwP//PRP-T vaccine, (1) a DT booster dose at the age of 5 years provoked a 150-fold increase in tetanus antitoxin GMT, (2) a high tetanus antitoxin GMT value was attained (GMT = 19.3 IU/mL) and (3) all children in this group had tetanus antitoxin titers > 1.0 IU/mL, so it may be concluded that all these children will still be protected against tetanus until at least the age of the next recommended booster dose (i.e. the age of 15 years). No differences in the occurrence of adverse events were observed between the groups who received the DTwP//PRP-T vaccine or the DTwP//Placebo vaccine, both vaccines being associated with events customarily attributable to DTwP (data not shown). Our results indicate (1) that the combination vaccine, DTwP//PRP-T, represents a safe and effective alternative for the existing uncombined vaccines and (2) that the long-term effect of interference between the components of future combination vaccines should be studied with subsequent booster doses, followed by the evaluation of persistence of antibodies over several years.