Induction of protective immunity in swine by immunization with live attenuated recombinant pseudorabies virus expressing the capsid precursor encoding regions of foot-and-mouth disease virus

Foot-and-mouth disease (FMD) causes morbidity to livestock and serious economic consequences to its associated industry and therefore it is necessary to develop a safe and efficient vaccine to prevent or control this disease. A recombinant live attenuated virus vaccine, designated PRV-P1, was generated by insertion of an expression cassette containing CMV promoter, FMDV P1 gene and SV 40 poly-A into the gG gene region of a live attenuated pseudorabies virus vaccine strain (TK⁻/gG⁻/LacZ⁺). To determine the induction of protective immunity, 16 FMDV and PRV seronegative white swine were randomly divided into four groups and immunized intramuscularly. The parental virus (TK⁻/gG⁻/LacZ⁺) was injected into three pigs, the recombinant virus PRV-P1 into five pigs and commercial FMD-inactivated vaccine into five pigs, with PBS (negative control) into three pigs. All animals were immunized again 4 weeks later to boost the immune response and challenged with virulent type O FMDV O/ES/2001 strain 4 weeks after the second immunization. Results showed PRV-P1 vaccinated pigs induced high-level neutralizing antibody response to both FMDV and PRV, and strong CTL response against FMD antigen activation. Three of five pigs were completely protected against challenge with FMDV, one pig minimally protected and the other one had increased protection but not complete. However, one pig vaccinated with commercial FMD vaccine developed constant pyrexia. Average levels of antibodies against non-structural 3ABC proteins were significantly lower and efficacy on inhibition of FMDV replication was much increased in swine vaccinated with PRV-P1 than those immunized with commercial FMD vaccine after FMDV challenge. Our results showed that the recombinant PRV-P1 can induce not only humoral and cell-mediated immune responses but also partial protection against FMDV challenge, making it a good candidate for future development of the FMD vaccine.     

A gE-negative bovine herpesvirus 1 vaccine strain is not re-excreted nor transmitted in an experimental cattle population after corticosteroid treatments

To study possible reactivation and to quantify subsequent transmission of a live gE-negative bovine herpesvirus 1 (BHV1) vaccine strain in cattle populations, four experiments were performed. Two groups of cattle were each tested twice for the possibility of reactivation. Inoculation with a gE-negative BHV1 vaccine was done either intramuscularly or intranasally and treatment with corticosteroids in an attempt to reactivate vaccine virus, was done after 6 or 11 weeks, and again after 6 months. To quantify transmission of vaccine virus following possible reactivation, each cattle was housed together with one susceptible contact-cattle. Contact-infections were monitored using virus shedding and antibody responses. After corticosteroid treatments, re-excretion of virus was never detected in cattle that had been inoculated with the gE-negative BHV1 vaccine strain. Contact cattle did not shed gE-negative BHV1, nor mounted any antibody response against BHV1. In contrast, positive control cattle, inoculated intranasally with wild-type BHV1, re-excreted virus in high titers in nasal fluids and transmitted the virus to contact cattle. Based on these results, the transmission ratio R(0) of the vaccine strain was zero. We concluded that it is highly unlikely that the live gE-negative BHV1 vaccine strain will be re-excreted after possible reactivation, and consequently, it is even less likely that reactivated vaccine virus will spread in the cattle population.     

Specific passive immunity reduces the excretion of glycoprotein E-negative bovine herpesvirus type 1 vaccine strain in calves

We investigated the excretion of either a glycoprotein E (gE)-negative bovine herpesvirus type 1 (BHV1) vaccine strain or a conventional modified-live vaccine strain in both na?ve and passively immunised calves. The replication of gE-negative strain was considerably reduced in the maternally immunised calves, in comparison with the non-immune calves. On the other hand, the excretion of the gE-positive conventional vaccine strain was not reduced and even seemed to be prolonged in the presence of maternal antibodies. These results suggest that BHV1 gE may play a role in virus survival in the presence of antibodies.     

Construction of a triple gene-deleted Chinese Pseudorabies virus variant and its efficacy study as a vaccine candidate on suckling piglets

New-emerging variants of Pseudorabies virus (PRV) compromise the protection provided by current vaccines and cause the death of all ages of vaccinated pigs since 2011. New vaccines based on current circulating PRV strain are needed to control the spread of disease since the variants are antigenically different from classical strains of virus. In this study, a TK/gE/gI triple gene-deleted PRV derived from current circulating field isolate was generated by using bacterial artificial chromosome techniques, and the rescued virus showed similar growth properties in vitro to its parent strain but reduced plaque size. To evaluate it as vaccine candidate, 9 day-old pigs were vaccinated and challenged with a virulent PRV variant. The results showed that vaccination can generate high level of protective gB-specific antibodies after vaccination and provide complete protection to the viral challenge. By contrast, the unvaccinated piglets all died within 6 days after viral challenge. Therefore, the TK/gE/gI triple gene-deleted PRV could be a promising vaccine candidate to control the wide spreading of PR variants in China.     

Novel pseudorabies virus variant with defects in TK,gE and gI protects growing pigs against lethal challenge

One of the distinct features of the emerging Chinese pseudorabies virus (PRV) variant is its ability to cause severe neurological signs and high mortality in growing pigs in Bartha-K61-vaccinated pig farms. Either single- or multiple-gene-deleted live vaccine candidates have been developed; however, none was evaluated thoroughly in growing pigs. Here, we generated rSMXΔgI/gEΔTK, an attenuated PRV variant with defects in TK, gI and gE genes. The growth kinetics of the attenuated virus was similar to the wild type (wt) strain. It was safe for 1-day-old piglets. Twenty one-day-old weaned pigs were immunized intramuscularly either with 10^6.0 TCID₅₀ of rSMXΔgI/gEΔTK or one dose of commercial Bartha-K61 vaccine, or with DMEM, and were challenged intranasally with 10^7.0 TCID₅₀ wt virus at 28 days post vaccination. rSMXΔgI/gEΔTK elicited higher level neutralization antibody against both PRV variant SMX and Bartha-K61 strain, while Bartha-K61 vaccine elicited lower neutralization activity of antibody against SMX. After challenge, all pigs in rSMXΔgI/gEΔTK group survived without any clinical signs, while unvaccinated group showed 100% mortality, and Bartha-K61 group showed severe respiratory symptoms and 3 out of 5 pigs exhibited severe neurological signs. Pigs in rSMXΔgI/gEΔTK group gained significantly higher body weight and diminished viral excretion titer and period, compared with Bartha-K61 group. Furthermore, the safety and efficacy of rSMXΔgI/gEΔTK was also evaluated in sheep and compared with local vaccine in growing pigs. These data suggest that the attenuated strain rSMXΔgI/gEΔTK is a promising live marker vaccine candidate for PR control in the context of emerging PRV variants.     

Bartha-k61 vaccine protects growing pigs against challenge with an emerging variant pseudorabies virus

Since late 2011, pseudorabies (PR) has resurfaced in many large pig farms, causing great economic loss for the swine industry in China. The PRV variant strain with high virulence and antigenic variation has been considered to be the main cause, and much attention has been focused on how to prevent and control the reoccurrence of this disease in China. In this study, two kinds of vaccination strategy were employed to evaluate the protective effects of Bartha-k61vaccine against both variant PRV (XJ5) and classical PRV (Ra) strain challenge. Humoral immunity response, clinical signs, survival rate, body weight, virus shedding and pathology were assessed in commercial pigs. The results showed that Bartha-k61vaccine, administered either once or twice, was effective against the PRV variant (XJ5) challenge, while no significant differences were observed between single and prime-boost vaccinated pigs. However, pigs vaccinated twice had better body weight gains than those vaccinated once, following challenge with the classical PRV strain (Ra) (p < 0.01). Therefore, the Bartha-k61 vaccine appears to be an effective vaccine to control the spread of PRV variants in China in the absence of new powerful candidate vaccines specific to these PRV strains.     

The influence of maternal immunity on the transmission of pseudorabies virus and on the effectiveness of vaccination

The purpose of this study was to investigate whether maternal immunity could prevent transmission of pseudorabies virus (PRV) among pigs, and whether it reduced the effectiveness of a single or double vaccination with regard to the transmission of PRV. In five experiments, the transmission of PRV, expressed as the reproduction ratio R, was compared in groups of pigs with maternal immunity and in groups of pigs without maternal immunity. Transmission of PRV among unvaccinated pigs with maternal immunity (R=0.2) was significantly lower than among pigs without maternal immunity (R=6.3). Furthermore, maternal immunity in young pigs prevented transmission of PRV, as R was significantly below one. In once-vaccinated groups, PRV spread extensively among pigs with maternal immunity (R=23), but did not spread extensively among pigs without maternal immunity (R=0.6). In twice-vaccinated groups, transmission of PRV among pigs with maternal immunity (R=0.6) did not differ significantly from the transmission of PRV among pigs without maternal immunity (R=0.3). Thus, a single vaccination of pigs with PRV strain 783 at 10 weeks of age, when they still possessed maternal immunity, seemed not sufficient to prevent transmission of PRV. Virus transmission could be reduced, however, if maternally immune pigs were vaccinated twice at 10 and 14 weeks of age.     

Sterilizing immunity against experimental Helicobacter pylori infection is challenge-strain dependent

The development of a murine model of Helicobacter pylori infection through serial in vivo passage of candidate strains has enabled a quantitative assessment of vaccine efficacy. In this study we compare infection with and protection against challenge from both CagA⁺ type I, and CagA⁻ type II in vivo adapted isolates. In vivo passage of a type II H. pylori isolate resulted in a highly infectious strain (X47-2AL), capable of reproducibly infecting mice to high density (10⁷ CFU/g of gastric tissue). Similarly adapted type I strains were found to colonize mice at a significantly lower level (10⁴–10⁵ CFU/g tissue). Mucosal immunization with recombinant urease (rUre) significantly protected animals against both types. Protection against X47-2AL was characterized by a ≥100-fold (or 2 log) reduction in bacterial density. However, the presence of a residual infection highlighted the inability to achieve sterilizing immunity against this strain. The level of protection appeared independent of challenge dose, and was stable for up to 6 months, all animals exhibiting a low-level residual infection that did not recrudesce with time. Similarly immunized mice challenged with isolates representing the residual infection were also protected, confirming that they did not represent a sub-population of H. pylori that could escape immunity. Immunization and challenge studies with type I adapted-isolates, demonstrated a similar 2–3 log reduction in the bacterial burden, but that in this instance resulted in sterilizing immunity. These results suggest varied specificity for the murine host by different Helicobacter strains that can influence the outcome of both infection and immunity.     

Association of HLA-DR14—DR52 with low responsiveness to hepatitis B vaccine in Chinese residents in Taiwan

To determine the HLA-linked immune response gene that controls low responsiveness to hepatitis B surface antigen (HBsAg), HLA typing was performed in 33 initial non-responders (male:female = 23:10, age 1.5–46 years) who had poor antibody response (anti-HBs < 10 mIU ml⁻¹) after four doses of plasma-derived hepatitis B vaccine. Of 33 initial non-responders, 26 received two additional doses of either the same vaccine (n = 18) or recombinant hepatitis B vaccine (n = 8) and returned for anti-HBs measurement. At 1 month after the sixth dose, anti-HBs was still <10 mIU ml⁻¹ in 20 cases and 10–20 mIU ml⁻¹ in three cases. Analysis of HLA antigen frequencies in these 23 ultimate low responders revealed that nine (39%) were positive for DR14, a statistically significant association of low responsiveness to hepatitis B vaccine with HLA-DR14. In addition, 26% of the ultimate low-responders were positive for DQ3, a frequency significantly lower than the expected rate in the general population. Among the nine ultimate low-responders with DR14, seven were heterozygous for this allele, while the other two cases had a single isolated DR14; and all nine were in association with DR52. These results suggest that a DR14-DR52 association, probably dominantly expressed, may be involved in the low immune responsiveness to hepatitis B vaccine of the Chinese population in Taiwan.     

A search for serologic correlates of immunity to Bordetella pertussis cough illnesses

In a pertussis vaccine efficacy trial in Germany we collected sera from vaccinees (DTaP or DTP) after the third and fourth doses of vaccine or at comparable time periods in DT vaccine recipients. In addition, sera were collected from a randomized sample of subjects in each vaccine group at approximately 3-month intervals from which antibody kinetic curves were constructed, which allowed us to estimate specific antibody values to pertussis toxin (PT), filamentous hemagglutinin (FHA), pertactin and fimbriae-2 at the time of exposure in the household setting. The imputed geometric mean antibody values to PT, pertactin and fimbriae-2 at the time of household exposure to Bordetella pertussis infection were higher (p < 0.07 or lower) in non-cases compared with cases. A multivariate (classification tree) analysis found that only pertactin and PT were significant in protection. Subjects with an imputed pertactin value of < 7 EU ml⁻¹ had a 67% (18/27) chance of infection regardless of the PT value. If the pertactin value was ≥ 7 EU ml⁻¹ and the PT value ≥ 66 EU ml⁻¹ all subjects were non-cases. If the pertactin value was ≥ 7 and the PT value was < 66 EU ml⁻¹ the predicted probability of being a case was 31% (15/49). Logistic regression analysis also found that high versus low pertactin values were associated with illness prevention following household exposure. In the presence of antibody to pertactin, PT and fimbriae-2, the additional presence of antibody to FHA did not contribute to protection. Our data support historical data indicating that agglutinating antibodies are associated with protection and also recent serologic correlates data and clinical efficacy data which indicate that multicomponent vaccines containing pertactin and fimbriae have better efficacy than PT or PT/FHA vaccines.     

Assessment of the immunogenicity and reactogenicity of a quadrivalent diphtheria, tetanus, acellular pertussis and hepatitis B (DTPa-HBV) vaccine administered in a single injection with Haemophilus influenzae type b conjugate vaccine, to infants at 2, 4 and 6 months of age

This double-blind, randomised study was performed to assess the immunogenicity and reactogenicity of three lots of a quadrivalent diphtheria-tetanus-acellular pertussis-hepatitis B vaccine (DTPa-HBV) co-administered with three lots of Haemophilus influenzae type b conjugate (Hib) vaccine in one injection, as a primary vaccination course in healthy infants at 2, 4 and 6 months of age. 269 infants (8–11 weeks of age) were randomly allocated to three groups to receive DTPa-HBV/Hib vaccines, concomitantly with oral polio vaccine. Blood samples for antibody determinations were taken before vaccination and 1 month after the third dose in 262 subjects. Local and general symptoms were recorded by parents on diary cards. All vaccinees had post-vaccination protective anti-D and anti-T (≥0.1 IU ml⁻¹) antibodies, and 98% had protective anti-HBs antibody titres (≥10 mIU ml⁻¹). There were no statistically significant differences between groups in post-vaccination anti-D, anti-T, anti-HBs antibody geometric mean titres (GMT), these being 3.49 IU ml⁻¹, 5.92 IU ml⁻¹ and 1109 mIU ml⁻¹, respectively. All subjects responded to three pertussis components, i.e. pertussis toxin (PT), filamentous haemagglutinin (FHA) and pertactin (PRN). Although statistically significant differences in GMTs of anti-PT, anti-FHA and anti-PRN were found between groups, these were not believed to be of any clinical relevance as the minimum GMTs were 60, 193 and 230 EL.U ml⁻¹ for anti-PT, anti-FHA and anti-PRN, respectively. There were no statistically significant differences in anti-PRP antibody GMT (4.05 μg ml⁻¹) between groups, 100% and 85% of subjects having titres ≥0.15 and 1.0 μg ml⁻¹, respectively. No symptoms were reported for one third of the subjects. Fever ( > 38°C) was reported after 16% of doses, with <1% having >39.5°C. Almost all local and general symptoms were mild or moderate, and lasted less than 48 h. No subject dropped out due to a severe adverse reaction. The administration of an experimental mix of DTPa-HBV and Hib vaccines in a single injection is safe, well-tolerated and immunogenic for all vaccine components.     

A gE-negative BHV1 vaccine virus strain cannot perpetuate in cattle populations

Three identical transmission experiments were successively performed to quantitatively evaluate the possible transmission of a gE-negative bovine herpesvirus 1 (BHV1) vaccine strain among cattle. After intranasal inoculation, the vaccine virus was excreted in high titers in nasal fluids. However, the vaccine virus was transmitted to only one sentinel in one experiment, and not to any of the 10 sentinel cattle in the other two experiments. Based on these observations, it can be concluded that the expected number of cases per vaccine-inoculated animal, i.e. the transmission ratio R(0) of the vaccine strain, is significantly below 1. The R(0) was estimated to be 0.14. After intramuscular inoculation, shedding of vaccine virus was not detected. Therefore, we concluded that it is highly unlikely that this live gE-negative BHV1 vaccine strain will perpetuate in the cattle population.     

Determination of the onset of the herd-immunity induced by the E2 sub-unit vaccine against classical swine fever virus

For a recently developed E2 subunit vaccine against classical swine fever (CSF), the reduction in transmission, at different moments after vaccination, was assessed by animal experiments and statistical calculations. Two experiments were performed to estimate the reproduction ratio R. Experiment 1 consisted of three groups and experiment 2 of two groups each of 10 pigs. In four of these groups, all pigs were vaccinated intramuscularly with the vaccine. The pigs in the fifth group remained unvaccinated (control group). After treatment, half of each group was intranasally inoculated with the virulent CSFV strain Brescia. In the vaccine groups, the following vaccination-challenge intervals were applied: 14, 14, 10, and 7 days, respectively. The occurrence of (contact-) infection was determined using the E(rns) ELISA. In the 7-days interval group and in the control group, virus transmission to all contact pigs occurred, indicating R1. Neither in the two 2-week interval groups nor in the 10-day interval group did contact-infections occur. Hence, the estimated R is less than one, which indicates that an epidemic would fade out. Therefore, the E2 subunit vaccine may be an efficacious tool in a control program during an outbreak of CSF as from 10 days after vaccination.     

Pseudorabies Virus Variant in Bartha-K61–Vaccinated Pigs,China, 2012

The widely used pseudorabies virus (PRV) Bartha-K61 vaccine has played a key role in the eradication of PRV. Since late 2011, however, a disease characterized by neurologic symptoms and a high number of deaths among newborn piglets has occurred among Bartha-K61–vaccinated pigs on many farms in China. Clinical samples from pigs on 15 farms in 6 provinces were examined. The PRV gE gene was detectable by PCR in all samples, and sequence analysis of the gE gene showed that all isolates belonged to a relatively independent cluster and contained 2 amino acid insertions. A PRV (named HeN1) was isolated and caused transitional fever in pigs. In protection assays, Bartha-K61 vaccine provided 100% protection against lethal challenge with SC (a classical PRV) but only 50% protection against 4 challenges with strain HeN1. The findings suggest that Bartha-K61 vaccine does not provide effective protection against PRV HeN1 infection.     

某院CRAB同源性及其生物膜形成能力分析

目的分析某院耐碳青霉烯鲍曼不动杆菌(CRAB)的耐药特性、主要流行克隆型及生物膜形成能力,为更好地防控CRAB感染提供参考。方法收集该院32株非重复CRAB菌株,采用全自动微生物分析系统进行药敏检测,结晶紫染色法检测CRAB生物膜形成能力,脉冲场凝胶电泳(PFGE)分析CRAB克隆多态性。结果 32株CRAB对四环素的耐药率最低(68.6%),对其他药物的耐药率均78%。PFGE同源性结果显示,32株CRAB可分为9个(A～I型)不同克隆型,每个克隆型包括1～9株,主要的流行克隆型是A型(9株)和E型(7株)。32株CRAB中,14株(43.8%)有生物膜形成能力且均为弱阳性;各克隆型间生物膜形成能力比较,差异无统计学意义(χ~2=6.636,P=0.659)。随着生物膜形成能力的增强,耐药率均有不同程度地升高,但产膜菌与非产膜菌两组间仅庆大霉素耐药率比较,差异具有统计学意义(χ~2=4.879,P0.05)。结论该院CRAB存在以A型和E型为主的不同克隆型传播,生物膜形成能力的增强能提高菌株的耐药性。     

Seroprevalence investigation and genetic analysis of pseudorabies virus within pig populations in Henan province of China during 2018–2019

In late 2011, the outbreak of pseudorabies (PR) occurred in Bartha-K61-vaccinated pig farms and spread rapidly to many provinces of China, causing substantial economic losses to the swine industry. A total of 4708 pig serum samples from Henan province during 2018–2019 were collected to screen for the presence of pseudorabies virus (PRV) gE-specific antibodies, and phylogenetic analysis based on the gE gene of PRV was performed. Of the 4708 serum samples tested, 30.14% (1419/4708) were seropositive for PRV antibodies, based on PRV gE-coated enzyme-linked immunosorbent assay (ELISA), with slaughterhouses having the highest seroprevalence. The seropositive rates of PRV also varied with the region and the season. Phylogenetic analysis showed that three PRV isolates from this study were clustered in an independent branch together with the Chinese variant PRV strains (after 2012), and had a closer genetic relationship with the Chinese variant PRV strains, but differed genetically from the 4 early Chinese PRV strains and 4 European-American strains. This study suggests that three PRV isolates may belong to PRV variants, and the development of a novel vaccine against PRV variants is particularly urgent.     

Recombination in vivo of pseudorabies vaccine strains to produce new virus strains

Herpesvirus suis (pseudorabies virus, PRV) has been the focus of intensive genetic engineering efforts and several effective genetically recombinant modified live virus PRV vaccines have resulted. The likelihood and consequences of complementation and/or genetic recombination in vivo between genetically engineered and conventionally derived vaccine strains of PRV are essentially unknown. In this study, two vaccine strains of PRV with complementary gene deletions were co-inoculated into sheep. It reports that avirulent vaccine strains of PRV (genetically engineered and conventionally attenuated) recombined in vivo, resulting in the production of a new and undesirable strain of PRV. The present study exemplifies the need for thorough assessment of genetically engineered micro-organisms in the animal environment.     

Enhanced immunogenicity of a recombinant genital warts vaccine adjuvanted with monophosphoryl lipid A

The regression of genital warts is believed to be a T-cell-mediated immune effect. We have sought to enhance the immunogenicity of a therapeutic vaccine for the treatment of genital warts with the use of the adjuvant monophosphoryl lipid A (MPL^®-immunostimulant), a detoxified form of the lipopolysaccharide (LPS) of Salmonella minnesota R595. The comparative immunogenicity and reactogenicity of a recombinant human papillomavirus type 6 (HPV6) L2E7 fusion protein in either aqueous, oil-in-water emulsions or Alhydrogel^® formulations containing MPL^® was evaluated. We conclude that the simple addition of MPL^® to the L2E7 fusion protein already adsorbed onto Alhydrogel^® preferentially enhances antigen specific in vitro T-cell proliferative responses, IFNγ production and in vivo delayed type hypersensitivity responses without increasing its reactogenicity.     

农村中学生领悟社会支持、自尊与主观幸福感的关系

目的 探讨农村中学生领悟社会支持、自尊与主观幸福感之间的关系。方法 采用问卷调查法, 对两所学校927名农村中学生施测领悟社会支持、自尊与主观幸福感量表, 分析影响主观幸福感的中介。结果 领悟社会支持和主观幸福感呈显著正相关(r=0.403);自尊与主观幸福感呈显著正相关(r=0.660)。领悟社会支持对自尊和主观幸福感的回归效应显著(R²=0.097, R²=0.163);自尊在领悟社会支持基础之上对主观幸福感的回归效应显著(R²=0.436)。结论 领悟社会支持、自尊影响主观幸福感;自尊是领悟社会支持和主观幸福感之间关系的中介。     

A combined liquid Hib (PRP-OMP), hepatitis B, diphtheria, tetanus and whole-cell pertussis vaccine: uncontrolled preliminary clinical trial of immunogenicity and reactogenicity

We have conducted a preliminary uncontrolled clinical trial of the immunogenicity and reactogenicity of a new fully liquid pentavalent combination vaccination which incorporates a diphtheria, tetanus and whole-cell pertussis vaccine with Hib (PRP-OMP) and hepatitis B vaccines. Forty-five infants received three doses of the pentavalent vaccination at 2, 4, and 6 months of age, and then a fourth dose at 18 months of age. Subjects were bled prior to each vaccination, and a month after the third and fourth vaccinations. A 7-day diary card was used to record subject temperatures and other systemic and local clinical signs after each vacination. After the third dose, 98% of subjects had anti-PRP titres above 1 μg ml⁻¹ (95%ci 88%, 100%). Following boosting, the geometric mean titre (GMT) rose a mean 27-fold (95%ci 19-fold, 38-fold) to 33 μg ml⁻¹, and all subjects' titres (lower bound of 95%ci 92%) exceeded 1 μg ml⁻¹. For hepatitis B antibody, there was a GMT of 100 mIU ml⁻¹ after the third dose, and 86% of infants (95%ci 73%, 95%) had antibody levels ≥ 10 mIU ml⁻¹. After the fourth dose, there was a mean 77-fold boost (95%ci 48-fold, 130-fold) to a GMT of 860 mIU ml⁻¹ and 95% (95%ci 84%, 99%) of subjects had titres ≥ 10 mIU ml⁻¹. Diphtheria, tetanus, and pertussis antibody levels were all at acceptable levels after the first three doses and again after the fourth vaccination. The pentavalent vaccine was well tolerated at all administration times, and had a minor reactogenicity profile similar to DTPw alone as reported in previous studies. This study has provided preliminary evidence for both the safety and immunogenicity of the pentavalent vaccine given as a course at 2, 4, 6 and 18 months.