Inclusion of a universal tetanus toxoid CD4(+) T cell epitope P2 significantly enhanced the immunogenicity of recombinant rotavirus ΔVP8* subunit parenteral vaccines.

Currently available live oral rotavirus vaccines, Rotarix(®) and RotaTeq(®), are highly efficacious in developed countries. However, the immunogenicity and efficacy of such vaccines in some developing countries are low. We reported previously that bacterially-expressed rotavirus ΔVP8* subunit vaccine candidates with P[8], P[4] or P[6] specificity elicited high-titer virus neutralizing antibodies in animals immunized intramuscularly. Of note was the finding that antibodies induced with the P[8]ΔVP8* vaccine neutralized both homotypic P[8] and heterotypic P[4] rotavirus strains to high titer. To further improve its vaccine potential, a tetanus toxoid universal CD4(+) T cell epitope P2 was introduced into P[8] or P[6]ΔVP8* construct. The resulting recombinant fusion proteins expressed in Escherichia coli were of high solubility and were produced with high yield. Two doses (10 or 20 µg/dose) of the P2-P[8]ΔVP8* vaccine or P2-P[6]ΔVP8* vaccine with aluminum phosphate adjuvant elicited significantly higher geometric mean homologous neutralizing antibody titers than the vaccines without P2 in intramuscularly immunized guinea pigs. Interestingly, high levels of neutralizing antibody responses induced in guinea pigs with 3 doses of the P2-P[8]ΔVP8* vaccine persisted for at least 6 months. Furthermore, in the gnotobiotic piglet challenge study, three intramuscular doses (50 µg/dose) of the P2-P[8]ΔVP8* vaccine with aluminum phosphate adjuvant significantly delayed the onset of diarrhea and significantly reduced the duration of diarrhea and the cumulative diarrhea score after oral challenge with virulent human rotavirus Wa (G1P[8]) strain. The P2-P[8]ΔVP8* vaccine induced serum virus neutralizing antibody and VP4-specific IgG antibody production prechallenge, and primed the pigs for higher antibody and intestinal and systemic virus-specific IFN-γ producing CD4(+) T cell responses postchallenge. These two subunit vaccines could be used at a minimum singly or preferably in bivalent formulation to provide antigenic coverage of most of the G types of global importance.

Re-examination of immune response and estimation of anti-Vi IgG protective threshold against typhoid fever-based on the efficacy trial of Vi conjugate in young children.

BACKGROUND: The capsular polysaccharide of Salmonella enterica serovar Typhi, Vi antigen, is an essential virulence factor and a protective antigen. Similar to other polysaccharide vaccines, the protective action of Vi, both to the polysaccharide alone or when presented as a conjugate, is mediated by serum IgG Vi antibodies. The evaluation of Vi capsular polysaccharide based vaccines to prevent typhoid fever would be significantly facilitated by the identification of a "protective level" of serum antibodies to Vi antigen. METHODS: The protective level of anti-Vi IgG against typhoid fever was derived from the protective efficacy and immune response of a Vi-rEPA conjugate vaccine efficacy trial. The estimation was derived by two methods: correlation of the percent efficacy and the antibody distribution profile in the vaccine group at a given period of observation, and use of the relative ratio of anti-Vi IgG levels between the vaccine and placebo groups greater or equal to the Relative Risk of typhoid fever used in the efficacy determination. RESULTS: Both methods predicted a similar range of a minimum protective level of anti-Vi IgG between 1.4 and 2.0µg/ml (short term threshold). When applying a protective threshold of 10µg/ml at 6 months post immunization, an IgG level in excess of 1.4µg/ml was achieved by 90% of children at 46 months post immunization, consistent with an 89% level of protection over the duration of the study. We thus suggest that the proportion of children with Vi IgG>10µg/ml (long term threshold) 6 months after immunization may reflect the proportion protected over at least a 4 year period. CONCLUSION: The current assignment of an anti-Vi IgG protective level may be of value when evaluating vaccine performance of future Vi conjugate vaccines.

Phase I clinical trial of O-acetylated pectin conjugate, a plant polysaccharide based typhoid vaccine.

BACKGROUND: Typhoid fever remains an important cause of morbidity and mortality in the developing countries. Vi capsular polysaccharide conjugate vaccine demonstrated safety and efficacy in young children in high endemic regions. A novel typhoid conjugate vaccine based on plant polysaccharide pectin was studied in a phase I trial. METHODS: Fruit pectin, having the same carbohydrate backbone structure as Vi, was purified from citrus peel and used as the polysaccharide source to prepare a semi-synthetic typhoid conjugate vaccine. Pectin was chemically O-acetylated (OAcPec) to antigenically resemble Vi and conjugated to carrier protein rEPA, a recombinant exoprotein A from Pseudomonas aeruginosa. 25 healthy volunteers, 18-45 years old, were injected once with OAcPec-rEPA. Safety and IgG antibodies reactive with Vi and pectin were analyzed. RESULTS: No vaccine associated serious adverse reaction was reported. Six weeks after the injection of OAcPec-rEPA, 64% of the volunteers elicited >4-fold rise of anti-Vi IgG. At 26 weeks the level declined, but the difference between the levels at 6 and 26 weeks are not statistically significant. There is a direct correlation between the level of anti-Vi IgG before and after the injection (R(2)=0.96). The anti-Vi IgG can be absorbed by Vi, but not by pectin. There was no corresponding increase of anti-pectin after the injection, indicating the antibody response to OAcPec-rEPA was specific to Vi. There is no Vi-rEPA data in US adults for comparison of immune responses. The OAcPec-rEPA elicited significantly less IgG anti-Vi in US adults than those by Vi-rEPA in Vietnamese adults. CONCLUSION: The O-acetylated pectin conjugate, a plant based typhoid vaccine, is safe and immunogenic in adult volunteers. ClinicalTrial.gov identifier: NCT00277147, NIH Protocol ID number: OH06-CH-0070, FDA BB Investigation New Drug (IND) number 6989.

Clinical Studies of Escherichia coli O157:H7 Conjugate Vaccines in Adults and Young Children.

Pediatric immunization has been the most effective measure to prevent and reduce the burden of infectious diseases in children. The recent inclusion of pneumococcal and meningococcal polysaccharide conjugates in infant immunization further reinforces their importance. Currently there is no human vaccine against enterohemorrhagic Escherichia coli (EHEC) infections. This review focuses on the human EHEC vaccine that has been studied clinically, in particular, the polysaccharide conjugate against E. coli O157. The surface polysaccharide antigen, O-specific polysaccharide, was linked to rEPA, recombinant exotoxin A of Pseudomonas aeruginosa. In adults and children 2 to 5 years old, O157-rEPA conjugates, shown to be safe, induced high levels of antilipopolysaccharide immunoglobulin G with bactericidal activities against E. coli O157, a functional bioassay that mimics the killing of inoculum in vivo. A similar construct using the B subunit of Shiga toxin (Stx) 1 as the carrier protein elicited both bactericidal and toxin-neutralizing antibodies in mice. So far there is no clinical study of Stx-based human vaccine. Passive immunization of Stx-specific antibodies with humanized, chimeric, or human monoclonal antibodies, produced in transgenic mice, showed promising data in animal models and offered high prospects. Demonstrations of their safety and effectiveness in treating hemolytic-uremic syndrome or patients with EHEC infections are under way, and results are much anticipated. For future development, other virulence factors such as the nontoxic Stx B subunit or intimin should be included, either as carrier protein in conjugates or as independent components. The additional antigens from O157 may provide broader coverage to non-O157 Stx-producing E. coli and facilitate both preventive and therapeutic treatment.

Development of Vi conjugate - a new generation of typhoid vaccine.

Typhoid fever remains to be a serious disease burden worldwide with an estimated annual incidence about 20 million. The licensed vaccines showed moderate protections and have multiple deficiencies. Most important of all, none of the licensed typhoid vaccines demonstrated protection for children under 5 years old. These limitations impeded successful implementation of typhoid vaccination programs. To improve immunogenicity Vi was conjugated to rEPA, a recombinant exoprotein A from Pseudomonas aeruginosa. Vi-rEPA showed higher and longer lasting anti-Vi IgG in adults and children than Vi alone in high endemic areas. In school-age children and adults, the immunity persisted more than 8 years. In a double-blind, placebo-controlled and randomized efficacy trial in 2- to 5-year-old children, Vi-rEPA conferred 89% protective efficacy against typhoid fever and the protection lasted at least 4 years. When given concomitantly with infant routine vaccines, Vi-rEPA was safe, immunogenic and showed no interference with the routine vaccines. Vi conjugate vaccine was also attempted and successfully demonstrated by several other laboratories and manufactures. Using either rEPA or different carrier proteins, such as diphtheria or tetanus toxoid, recombinant diphtheria toxin (CRM197), the Vi conjugates synthesized was significantly more immunogenic than Vi alone. Recently, two Vi-tetanus toxoid conjugates were licensed in India for all ages, starts as young as 3 month old. This new generation of typhoid vaccine opens up a new era for typhoid prevention and elimination.

Immunochemical characterization of synthetic hexa-, octa- and decasaccharide conjugate vaccines for Vibrio cholerae O:1 serotype Ogawa with emphasis on antigenic density and chain length.

Cholera remains to be a global health problem without suitable vaccines for endemic control or outbreak relief. Here we describe a new parenteral vaccine based on neoglyco-conjugate of synthetic fragments of O-specific polysaccharide (O-SP) of Vibrio cholerae O1, serotype Ogawa. Hexa-, octa- and decasaccharides of the O-SP with carboxylic acid at the reducing end were chemically synthesized and conjugated to tetanus toxoid (TT). The conjugates prepared by a novel linking scheme consisted of 17-atom linker of hydrazide and alkyl bonds elicited robust serum IgG anti-LPS responses with vibriocidal activities in mice. There is a length dependence in immune response with decasaccharide conjugates elicited the highest anti-LPS IgG. There seems to be an indication that regardless of the carbohydrate chain length, a molar ratio of 230 ± 10 monosaccharide units per TT induced high antibody response. The conjugates also elicited cross-reactive antibodies to serotype Inaba. The formulation of the proposed cholera conjugate vaccine, similar to other licensed polysaccharide vaccine, is suitable for children immunization. A parenteral cholera vaccine could overcome the diminishing immunogenicity in most of oral vaccines due to the gastrointestinal complexity and environmental enteropathy in children living in impoverished environment and could be considered for global cholera immunization.

A human IgG anti-Vi reference for Salmonella typhi with weight-based antibody units assigned.

Recent data showing the high incidence of typhoid fever in young children, the demonstration of safety and efficacy of a Vi conjugate for this age group, the safety and similar immunogenicity in infants when administrated concurrently with EPI vaccines, together with the interests of manufacturers and investigators in studying such conjugate vaccines prompted us to prepare a human IgG anti-Vi standard to facilitate this work. Volunteers were injected with an investigational Vi-recombinant Pseudomonas aeruginosa exoprotein A (Vi-rEPA) conjugate vaccine. Plasmas with the highest levels of IgG anti-Vi were pooled. The IgG anti-Vi content of this preparation, assayed by precipitin analysis with purified Vi, was 33 µg/ml. Accordingly, the estimated IgG anti-Vi protective level of 3.5 ELISA unit/ml, derived from our efficacy trial of Vi-rEPA in 2-5 years old children, is equivalent to 4.3 µg/ml. This reagent is suitable for comparison of immune response of Vi conjugate vaccines or for other purposes requiring anti-Vi measurement.

Construction and characterization of human rotavirus recombinant VP8* subunit parenteral vaccine candidates.

Two currently licensed live oral rotavirus vaccines (Rotarix® and RotaTeq®) are highly efficacious against severe rotavirus diarrhea. However, the efficacy of such vaccines in selected low-income African and Asian countries is much lower than that in middle or high-income countries. Additionally, these two vaccines have recently been associated with rare case of intussusception in vaccinated infants. We developed a novel recombinant subunit parenteral rotavirus vaccine which may be more effective in low-income countries and also avert the potential problem of intussusception. Truncated recombinant VP8* (ΔVP8*) protein of human rotavirus strain Wa P[8], DS-1 P[4] or 1076 P[6] expressed in Escherichia coli was highly soluble and was generated in high yield. Guinea pigs hyperimmunized intramuscularly with each of the ΔVP8* proteins (i.e., P[8], P[4] or P[6]) developed high levels of homotypic as well as variable levels of heterotypic neutralizing antibodies. Moreover, the selected ΔVP8* proteins when administered to mice at a clinically relevant dosage, route and schedule, elicited high levels of serum anti-VP8* IgG and/or neutralizing antibodies. Our data indicated that the ΔVP8* proteins may be a plausible additional candidate as new parenteral rotavirus vaccines.

Early-life and contemporaneous nutritional and environmental predictors of antibody response to vaccination in young Gambian adults.

Recent research links nutritional exposures early in life with alterations in functional immunity that persist beyond childhood. Here we investigate predictors of antibody response to polysaccharide vaccines in a cohort of Gambian adults with detailed records from birth and early infancy available. 320 adults were given a single dose of a Vi polysaccharide vaccine for Salmonella typhi and a 23-valent capsular polysaccharide pneumococcal vaccine. Anti-Vi antibody levels and antibodies against 4 pneumococcal serotypes (1, 5, 14 and 23F) were measured in serum samples collected at baseline and then 14 days following vaccination and compared to data available from birth and early infancy. Post-vaccination antibody titres to serotype 14 of the pneumococcal vaccine were negatively associated with rate of growth from birth to three months of age, infant weight at 12 months of age and season of birth, but no other associations were observed with early-life exposures. The strongest predictor of antibody levels was pre-vaccination antibody titres, with adult height and serum neopterin levels at time of vaccination also implicated. The current study does not support the hypothesis that nutritional exposures early in life consistently compromise antibody response to polysaccharide vaccines administered in young adulthood.

A bivalent vaccine to protect against Streptococcus pneumoniae and Salmonella typhi.

Pneumococcal and Salmonella typhi infections are two major diseases for children in developing countries. For typhoid fever, licensed Vi polysaccharide vaccines are ineffective in children <2-year old. While investigational Vi conjugate vaccines have been shown effective in clinical trials, they are currently only available to restricted areas. Pneumococcal capsular polysaccharide conjugate vaccines are highly effective in children, but suffer from some limitations including cost and limited serotype coverage. We have previously shown that a fusion conjugate vaccine, consisting of pneumococcal fusion protein PsaA and pneumolysoid (PdT) conjugated to a polysaccharide, results in enhanced antibody and CD4+ Th17 cell responses as well as protection against pneumococcal colonization and disease in mice. Here we applied this approach to develop a bivalent vaccine against pneumococcus and S. typhi. Two species-conserved pneumococcal antigens (SP1572 or SP2070) were fused to the nonhemolytic pneumolysoid PdT. SP1572-PdT was then conjugated to Vi polysaccharide and SP2070-PdT was conjugated to the pneumococcal cell wall polysaccharide (CWPS; also conserved). Mice immunized with this bivalent conjugate were protected against pneumococcal colonization and sepsis challenges, and made anti-Vi antibody concentrations higher by 40-fold compared to mice that received equimolar mixtures of the antigens. An enhanced killing of Vi-bearing Salmonellae in vitro was demonstrated from plasma of mice that received the fusion conjugate but not the mixture of antigens. Our results support further evaluation of this bivalent immunogen for the prevention of pneumococcal colonization and disease, and of typhoid fever.

The Vi conjugate typhoid vaccine is safe, elicits protective levels of IgG anti-Vi, and is compatible with routine infant vaccines.

Typhoid fever remains a serious problem in developing countries. Current vaccines are licensed for individuals who are 5 years old or older. A conjugate of the capsular polysaccharide (CP) of Salmonella enterica serovar Typhi (Vi) bound to recombinant exoprotein A of Pseudomonas aeruginosa (Vi-rEPA) enhanced Vi immunogenicity and protected 2- to 5-year-olds in Vietnam. In this study, Vi-rEPA was evaluated for use in infants. A total of 301 full-term Vietnamese infants received Expanded Program on Immunization (EPI) vaccines alone or with Vi-rEPA or Haemophilus influenzae type b-tetanus toxoid conjugate (Hib-TT) at 2, 4, and 6 months and Vi-rEPA or Hib-TT alone at 12 months. Infants were visited 6, 24, and 48 h after each injection to monitor adverse reactions. Maternal, cord, and infant sera were assayed for IgG anti-Vi and for IgG antibodies to Hib CP and the diphtheria, tetanus, and pertussis toxins at 7, 12, and 13 months. No vaccine-related serious adverse reactions occurred. In the Vi-rEPA group, the IgG anti-Vi geometric mean (GM) increased from the cord level of 0.66 to 17.4 enzyme-linked immunosorbent assay units (EU) at 7 months, declined to 4.76 EU at 12 months, and increased to 50.1 EU 1 month after the 4th dose (95% of infants had levels of ≥ 3.5 EU, the estimated protective level). Controls had no increase of the IgG anti-Vi GM. Infants with cord anti-Vi levels of <3.5 EU responded with significantly higher IgG anti-Vi levels than those with levels of ≥ 3.5 EU. Anti-diphtheria, -tetanus, and -pertussis toxin levels were similar in all groups. Vi-rEPA was safe, induced protective anti-Vi levels, and was compatible with EPI vaccines, and it can be used in infants. High cord IgG anti-Vi levels partially suppressed infant responses to Vi-rEPA.

Physical and chemical characterization and immunologic properties of Salmonella enterica serovar typhi capsular polysaccharide-diphtheria toxoid conjugates.

Typhoid fever remains a serious public health problem in developing countries, especially among young children. Recent studies showed more than 50% of typhoid cases are in children under 5 years old. Licensed vaccines, such as Salmonella enterica serovar Typhi capsular Vi, did not confer protection against typhoid fever for this age group. Vi conjugate, prepared by binding Vi to Pseudomonas aeruginosa recombinant exoprotein A (rEPA), induces protective levels of antibody at as young as 2 years old. Because of the lack of regulatory precedent for rEPA in licensing vaccines, we employed diphtheria toxoid (DT) as the carrier protein to accommodate accessibility in developing countries. Five lots of Vi-DT conjugates were prepared using adipic acid dihydrazide (ADH) as the linker. All 5 lots showed consistency in their physical and chemical characteristics and final yields. These Vi-DT conjugates elicited levels of IgG anti-Vi in young mice significantly higher than those in mice injected with Vi alone and induced a booster response upon reinjection. This booster effect was absent if the Vi replaced one of the two conjugate injections. Vi-DT was stable under repeated freeze-thaw (20 cycles). We plan to perform clinical evaluation of the safety and immunogenicity of Vi-DT when added to the infant combination vaccines.

Revaccination does not improve an observed deficit in antibody responses in Pakistani adults born of a lower birth weight.

We have previously shown that the generation of antibodies to a polysaccharide vaccine (Typhim Vi) is compromised in Pakistani adults born of a lower birth weight. To assess whether this represents a true B-cell-dependent deficit, we revaccinated subjects with a second dose of the same vaccine and with a polysaccharide-protein conjugate vaccine to a different polysaccharide antigen (conjugated Haemophilus influenzae type b (Hib) vaccine). Anti-Vi IgG levels remained positively correlated with birth weight (p=0.0284) but no associations were observed between anti-Hib IgG levels and size at birth. These findings indicate that small size at birth results in a poor antibody response to vaccination with a polysaccharide antigen vaccine in adulthood, even following a second dose of the vaccine. No such association was observed in response to a polysaccharide-protein conjugate vaccine indicating an early-life programming effect on the generation of antibodies during a B-cell-dependent immune response.

Safety and immunogenicity of Escherichia coli O157 O-specific polysaccharide conjugate vaccine in 2-5-year-old children.

BACKGROUND: Escherichia coli O157:H7 causes severe enteritis and hemolytic-uremic syndrome, mostly in young children and older adults. Similar to the case with Shigella, serum IgG against the O-specific polysaccharide of E. coli O157:H7 may confer immunity by lysing the inoculum in the intestine. A phase 1 trial in adults showed that a vaccine of E. coli O157:H7 O-specific polysaccharide conjugated to recombinant exotoxin A of Pseudomonas aeruginosa (O157-rEPA) was safe and immunogenic. METHODS: A phase 2 trial of the O157-rEPA vaccine was conducted in 49 children 2-5 years old who were divided randomly into groups receiving 1 or 2 doses of vaccine. Adverse reactions were monitored. Serum IgG lipopolysaccharide (LPS) antibodies were determined. RESULTS: No significant adverse reactions were observed. At 1 week after the first dose was administered, most children (81%) responded with a >4-fold increase in serum IgG LPS antibodies. At 6 weeks after the first dose was administered, all children responded with a >8-fold increase; a second dose did not elicit a booster response. At 26 weeks after the first dose was administered, the geometric mean titer of serum IgG LPS antibodies was ~20-fold higher than was the prevaccination titer. These serum samples had high titers of bactericidal activity that were correlated roughly with serum IgG LPS antibody titers (r = .78). CONCLUSIONS: The O157-rEPA vaccine was safe and immunogenic in young children. A phase 3 trial of the administration of this conjugate vaccine concurrently with routine immunization in infants is planned.

Effect of dosage on immunogenicity of a Vi conjugate vaccine injected twice into 2- to 5-year-old Vietnamese children.

In a double-blind, randomized, and placebo-controlled previous trial, the efficacy of Vi-rEPA for typhoid fever in 2- to 5-year-olds was 89.0% for 46 months. Vi-rEPA contained 25 microg of Vi and induced a greater-than-eightfold rise in immunoglobulin G (IgG) anti-Vi in all of the vaccinees tested. In this investigation, we conducted a dosage-immunogenicity study of 5, 12.5, and 25 microg of Vi-rEPA in this age group. Two doses of Vi-rEPA were injected 6 weeks apart. Blood samples were taken before and at 10 weeks (4 weeks after the second injection) and 1 year later. All postimmunization geometric mean (GM) levels were higher than the preimmune levels (P < 0.0001). At 10 weeks, the GM IgG anti-Vi level elicited by 25 microg (102 EU/ml) was higher than those elicited by 12.5 microg (74.7 EU/ml) and 5 microg (43 EU/ml) (P < 0.004): all of the children had > or = 3.52 EU/ml (estimated minimum protective level). One year later, the levels declined about sevenfold (13.3 and 11.3 versus 6.43 EU/ml, P < 0.0001) but remained significantly higher than the preimmune levels (P < 0.0001), and >96% of the children had a greater-than-eightfold rise. This study also confirmed the safety and consistent immunogenicity of the four lots of Vi-rEPA used in this and previous trials.

Birth weight predicts response to vaccination in adults born in an urban slum in Lahore, Pakistan.

BACKGROUND: Substantial evidence exists linking small size at birth to later-life susceptibility to chronic disease. Evidence is also emerging that some components of immune function may be programmed in early life. However, this evidence is limited and requires confirmation. OBJECTIVE: We investigated the association between size at birth and response to vaccination in a cohort of 257 adults (mean age: 29.4 y; 146 men) born in an urban slum in Lahore, Pakistan, during 1964-1978. DESIGN: A single dose of Vi polysaccharide vaccine for Salmonella typhi and 2 doses of rabies vaccine were given to each subject. Antibody titers were measured in prevaccination serum samples (Vi) and in postvaccination samples (Vi and rabies). RESULTS: The mean birth weight of the subjects was 3.24 kg; 14% of the subjects had low birth weights (<2.5 kg). Vaccine responses were not consistently associated with contemporary variables (month of study, sex, current age, or indicators of wealth). Response to typhoid vaccination was positively related to birth weight (anti-Vi immunoglobulin G: r = 0.138, P = 0.031; anti-Vi immunoglobulin M: r = 0.197, P = 0.034). Response to the rabies vaccine was not significantly associated with birth weight. CONCLUSIONS: These findings add to a growing body of evidence suggesting that components of the immune system may be permanently programmed by events in early life. The contrasting effects on typhoid and rabies responses suggest that antibody generation to polysaccharide antigens, which have greater B cell involvement, is compromised by fetal growth retardation.