Intranasal immunization with genetically detoxified diphtheria toxin induces T cell responses in humans: enhancement of Th2 responses and toxin-neutralizing antibodies by formulation with chitosan

We previously reported that intranasal immunization with a non-toxic mutant cross-reacting material (CRM)197 of diphtheria toxin, formulated with chitosan, generated protective neutralizing antibodies in mice and guinea pigs. Furthermore, we demonstrated that intranasal delivery of a powder formulation of the CRM197-based vaccine was well tolerated and significantly boosted antibody responses in adult volunteers. Here we report that intranasal booster immunization with CRM197 alone or with chitosan induced systemic T cell responses. We addressed for the first time the induction of T cell subtypes following intranasal vaccination in humans. Intranasal vaccination with CRM197, like parenteral immunization with a conventional diphtheria toxoid vaccine, enhanced antigen-specific IFN-gamma production. However, formulation of the nasal diphtheria vaccine with chitosan significantly augmented Th2-type responses, which correlated with protective levels of toxin-neutralizing antibodies in intranasally boosted individuals. The results suggest that vaccines capable of inducing strong Th2-type responses, such as CRM197 formulated with chitosan, have potential for the development of a protective mucosal vaccine against diphtheria in humans. Furthermore, our findings demonstrate that mucosal subunit vaccines with appropriate delivery systems have considerable potential for booster immunization of adults.     

Safety and immunogenicity of CRM197-conjugated pneumococcal-meningococcal C combination vaccine (9vPnC-MnCC) whether given in two or three primary doses

This randomized trial compares safety and immunogenicity when vaccinating infants with a pneumococcal-meningococcal conjugate vaccine in two doses vs. three doses. Infants (N=223) received 9vPnC-MnCC (CRM197-conjugated pneumococcal serotypes 1, 4, 5, 6B, 9V, 14, 18C, 19F, 23F and meningococcal C polysaccharides) either at 3 and 5 or 3, 4 and 5 months and a booster with either 9vPnC-MnCC or 23-valent pneumococcal-polysaccharide vaccine (23vPPS) and CRM197-MnCC, at 12 months. Safety was monitored and IgG measured at 3, 6, 12 and 13 months in all subjects and serum bactericidal activity (SBA) in half. The 9vPnC-MnCC vaccine was safe and induced significant IgG to all components. Three doses induced higher antibody GMCs (geometric mean concentrations) at 6 months to seven of nine pneumococcal serotypes. This was most significant for 6B and 23F (p<0.001), that also showed lower rate of responders>0.35 (6B, 23F) and >0.5 microg/mL (6B). Antibody GMCs remained lower following 9vPnC-MnCC booster in subjects primed with two doses although only significant for serotype 18C. Significant memory responses were observed 1 week after the 23vPPS toddler dose. MnCC-IgG GMC was lower after two doses, however with comparable SBA. This study shows that the 9vPnC-MnCC vaccine is safe and induces successful immunological memory, whether given in two or three primary doses.     

Induction of immunological memory in UK infants by a meningococcal A/C conjugate vaccine

The induction of immunological memory to serogroup A and C polysaccharides in UK infants immunized with three doses of a meningococcal A/C oligosaccharide CRM197 conjugate vaccine was investigated. Forty UK infants vaccinated previously with three doses of a meningococcal A/C oligosaccharide-CRM197 conjugate vaccine at 2, 3 and 4 months of age, were revaccinated at a mean age of 145.6 weeks with either a 10 or 50 microg dose of licensed meningococcal A/C polysaccharide vaccine. Serogroup-specific antibody and serum bactericidal antibody (SBA) responses were measured by enzyme-linked immunosorbent assay and serum bactericidal assays, respectively. Following challenge, anti-serogroup A and C polysaccharide antibody levels rose from pre-booster geometric mean concentrations (GMC) of 3.1 and 2.1 microg/ml respectively to 19.6 and 21.0 microg/ml 1 month post-booster. Serum bactericidal antibody geometric mean titres (GMTs) for serogroups A and C increased 156- and 113-fold from 2.1 and 7.1 pre-booster respectively to 327.4 and 800.7 post-booster. A serogroup A control group of 45 children received a 10 microg dose of licensed meningococcal A/C polysaccharide vaccine (with no prior history of serogroup A vaccination) had serogroup A SBA GMTs of 2.3 pre-vaccination rising to 8 post-vaccination with corresponding GMCs of 0.8 and 10.8 microg/ml. These rises in SBA following serogroup A/C conjugate vaccination are indicative of immunological priming.     

A clinical trial examining the effect of increased total CRM(197) carrier protein dose on the antibody response to Haemophilus influenzae type b CRM(197) conjugate vaccine

CRM(197) is a carrier protein in certain conjugate vaccines. When multiple conjugate vaccines with the same carrier protein are administered simultaneously, reduced response to vaccines and/or antigens related to the carrier protein may occur. This study examined responses of infants who, in addition to diphtheria toxoid/tetanus toxoid/acellular pertussis vaccine (DTaP) received either diphtheria CRM(197)-based Haemophilus influenzae type b conjugate vaccine (HbOC) or HbOC and a diphtheria CRM(197)-based combination 9-valent pneumococcal conjugate vaccine/meningococcal group C conjugate vaccine. Administration of conjugate vaccines with CRM(197) carrier protein load >50 microg did not reduce response to CRM(197) conjugate vaccines or immunogenicity to immunologically cross-reactive diphtheria toxoid.     

Effect of monophosphoryl lipid A (MPL) on T-helper cells when administered as an adjuvant with pneumocococcal-CRM197 conjugate vaccine in healthy toddlers

As new vaccines are developed, novel adjuvants may play an important role in eliciting an effective immune response. We evaluated the safety and adjuvant properties of monophosphoryl lipid A (MPL in 129 healthy toddlers immunized with two doses of nine-valent pneumococcal-CRM(197) protein conjugate vaccine (PCV9) combined with 10, 25, or 50 micro g of MPL with or without alum (AlPO(4)). Vaccine-specific humoral and cell-mediated responses were examined following the second dose of study vaccine. All doses of MPL were well-tolerated and a dose-dependent effect of MPL on specific cellular responses was observed. The 10 micro g MPL dose significantly enhanced CRM(197)-specific T-cell proliferation (P=0.02) and interferon-gamma (INF-gamma) production (P=0.009) compared to responses of controls who received PCV9 with AlPO(4). In contrast, CRM(197)-specific T-cell proliferation and interferon-gamma production of the 50 micro g MPL/AlPO(4) group were decreased when compared to controls although these differences did not reach statistical significance. IL-5 and IL-13 responses after immunization showed a similar pattern with increased production in the 10 micro g MPL group and decreased production in the 50 micro g MPL/AlPO(4) group compared to controls. There were no differences in serum IgG antibody concentrations to the nine vaccine pneumococcal capsular polysaccharides and carrier protein between the MPL-containing and control vaccine groups. These findings demonstrate a dose-dependent effect of MPL on T-helper cell type 1 (TH-1) responses to the carrier protein and also suggest an effect on T-helper cell type 2 (TH-2) responses.     

Antibody response and persistence in volunteers following immunization with varying dosages of a trivalent surface antigen influenza virus vaccine

The serum antibody responses and 50% protective levels (PL50) of antibody were determined, using the SRH test, at one and twelve months post-vaccination in a group of student volunteers immunized with one of three dosages of a trivalent surface-antigen influenza virus vaccine, or with placebo. It was found that, for the H3, H1 and B haemagglutinin components present in the vaccine, a dose of 6 micrograms HA elicited high serum antibody responses at one month post-immunization. High mean antibody levels and a high incidence of volunteers with PL50 values of antibody against each of the HA components of the vaccine remained in the volunteer group twelve months later. The results are discussed in relation to the vaccine dosage used and the nature of the population immunized.     

Antibody response and persistence in volunteers following immunization with varying dosages of a trivalent surface antigen influenza virus vaccine.

The serum antibody responses and 50% protective levels (PL50) of antibody were determined, using the SRH test, at one and twelve months post-vaccination in a group of student volunteers immunized with one of three dosages of a trivalent surface-antigen influenza virus vaccine, or with placebo. It was found that, for the H3, H1 and B haemagglutinin components present in the vaccine, a dose of 6 micrograms HA elicited high serum antibody responses at one month post-immunization. High mean antibody levels and a high incidence of volunteers with PL50 values of antibody against each of the HA components of the vaccine remained in the volunteer group twelve months later. The results are discussed in relation to the vaccine dosage used and the nature of the population immunized.     

Efficacy trial of a parenteral gonococcal pilus vaccine in men.

A randomized, placebo-controlled, double-blind efficacy trial of a purified gonococcal pilus vaccine composed of a single pilus type was tested in 3123 men and 127 women volunteers. Either 100 micrograms of vaccine or a placebo was given intradermally on day 1 and day 14. Each group was evenly matched with respect to age, sex, prior history of a sexually transmitted disease, sexual exposure during the study and attrition from the study. None of the women volunteers acquired gonorrhoea during the trial. In the male volunteers, 108 vaccine and 102 placebo recipients acquired gonorrhoea 15 days or later after the initial immunization. Vaccines developed a sustained ELISA antibody response to homologous and heterologous pili, but the latter titres were approximately 40% as high as the homologous pilus antibody rises. There were, however, no increases in inhibition of attachment antibody (IEA) titres. Local antibodies (semen) against homologous and heterologous strains were also elicited (ELISA). The vaccine was safe and did not alter the clinical expression of disease. This gonococcal pilus vaccine composed of a single pilus type failed to protect men against gonococcal urethritis.     

Differences in the avidity of antibodies evoked by four different pneumococcal conjugate vaccines in early childhood

Avidity of antibodies to Streptococcus pneumoniae type 6B, 14, 19F and 23F polysaccharides (PS) evoked by four different pneumococcal conjugate vaccines was compared. Infants were primed with pneumococcal PS conjugated to the variant diphtheria toxin CRM197 (PncCRM), diphtheria toxoid (PncD), tetanus toxoid (PncT) or meningococcal protein complex (PncOMPC) and boosted with the homologous conjugate or PS vaccine. No booster was given to children in the PncOMPC group. Relative antibody avidity was measured by thiocyanate EIA. No vaccine specific differences were found in avidity of anti-14 or -19F antibodies. By contrast, antibody avidity to 6B and 23F differed significantly between the vaccine groups, PncCRM and PncT inducing antibodies of highest avidity.     

Effect of vaccination with 3 recombinant asexual-stage malaria antigens on initial growth rates of Plasmodium falciparum in non-immune volunteers

A placebo controlled, randomised, double blind trial was conducted in human volunteers to test a mixture of three recombinant Plasmodium falciparum blood stage antigens for its ability to reduce the initial growth rates of parasites. The vaccine contained recombinant MSP2 (3D7 allele), a portion of MSP1 (190LCS.T3) and part of the RESA antigen (C terminal 771 amino acids) in the Montanide ISA 720 adjuvant (SEPPIC). Twelve volunteers received two doses of the vaccine, 6 weeks apart. The five participants in the placebo group received an equivalent volume of the adjuvant emulsion using the same schedule. Antibody responses were low, as has been reported in earlier studies with this combination, while T cell responses were stronger. All the volunteers were challenged with approximately 140 ring infected red cells of the 3D7 cloned line, 4 weeks after the second dose. Parasitaemia was determined once daily from day 4 using a sensitive and quantitative PCR assay. All the volunteers were infected and were treated on day 8, before any developed symptoms. There was no significant difference in initial parasite growth rates between the verum and placebo groups, nor was there any significant correlation between parasite growth rates and any of the measured immunological responses. These results suggest that the formulation tested in this trial did not generate immune responses that were strong enough to reduce parasite growth in naive volunteers.     

A mucosal vaccine against diphtheria: formulation of cross reacting material (CRM(197)) of diphtheria toxin with chitosan enhances local and systemic antibody and Th2 responses following nasal delivery

The development of new generation vaccines against diphtheria is dependent on the identification of antigens and routes of immunization that are capable of stimulating immune responses similar to, or greater than, those obtained with the parenterally-delivered toxoid vaccine, while reducing the adverse effects that have been associated with the traditional vaccine. In this study, we examined the cellular and humoral immune responses in mice generated after both parenteral and mucosal immunizations with cross-reacting material (CRM(197)) of diphtheria toxin. We found that both native and mildly formaldehyde-treated CRM(197) and conventional diphtheria toxoid (DT) induced mixed Th1/Th2 responses and similar levels of anti-DT serum IgG following parenteral immunization. In contrast, CRM(197) preparations were poorly immunogenic when administered intranasally in solution. However, formulation of the antigens with chitosan significantly enhanced their immunogenicity, inducing high levels of antigen-specific IgG, secretory IgA, toxin-neutralizing antibodies and T cell responses, predominately of Th2 subtype. Furthermore, intranasal immunization with CRM(197) and chitosan induced protective antibodies against the toxin in a guinea pig passive challenge model. We also found that priming parenterally with DT in alum and boosting intranasally with CRM(197) was a very effective method of immunization in mice, capable of inducing high levels of anti-DT IgG and neutralizing antibodies in the serum and secretory IgA in the respiratory tract. Our findings suggest that boosting intranasally with CRM(197) antigen may be very effective in adolescents or adults who have previously been parenterally immunized with a conventional diphtheria toxoid vaccine.     

Immune responses to an inactive vaccine against American cutaneous leishmaniasis together with granulocyte-macrophage colony-stimulating factor

The immunological response in healthy subjects to a crude leishmania antigen vaccine (Leishvacin) plus rhGM-CSF without prior Montenegro (DTH) skin testing was evaluated. Fifty-six healthy volunteers received vaccine plus either placebo or rhGM-CSF at day 0, followed by either a vaccine booster or placebo at day 21. IFN-gamma and IL-5 levels were significantly enhanced by day 21. The adjuvant group had a higher percentage of individuals with a significant response to vaccination than the corresponding placebo group. Eighty-six percent of all volunteers were DTH-positive by day 42. Leishvacin is capable of sensitizing lymphocytes from individuals not previously exposed to leishmania antigen. Use of rhGM-CSF enhanced the immune response, indicating that it may improve immunological response to the vaccine.     

Pre-clinical evaluation of a 15-valent pneumococcal conjugate vaccine (PCV15-CRM197) in an infant-rhesus monkey immunogenicity model

The incidence of invasive pneumococcal disease (IPD), caused by the approximately 91 serotypes of Streptococcus pneumoniae (PN), varies geographically and temporally as a result of changing epidemiology and vaccination patterns as well as due to regional measurement differences. Prevnar^® (Pfizer), the first licensed pneumococcal conjugate vaccine (PCV), comprises polysaccharides (PS) from 7 serotypes conjugated to the mutant diphtheria toxin carrier protein, CRM197. In the United States and elsewhere, this vaccine has been highly efficacious in reducing the incidence of IPD caused by vaccine serotypes, however, the incidence of non-vaccine serotypes (e.g., 19A, 22F, and 33F) has increased, resulting in the need for vaccines with higher valencies. In response, 10- and 13-valent PCVs have recently been licensed. To further increase serotype coverage, we have developed a 15-valent PCV containing PS from serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 22F, 23F and 33F conjugated to CRM197 and formulated on aluminum phosphate adjuvant. Vaccine immunogenicity was evaluated in infant rhesus monkeys since they, like human infants, respond poorly to unconjugated PN PS. Infant (2-3 month old) rhesus monkeys were vaccinated three times with PCV-15 or Prevnar^® at 2 month intervals, and serotype-specific IgG antibodies were measured using a multiarray electrochemiluminescence (ECL) assay. The results indicate that antibody responses to PCV-15 and Prevnar^® were comparable for the 7 common serotypes and that post-vaccination responses to PCV-15 were >10-fold higher than baseline for the 8 additional serotypes.     

A randomised, double-blind, controlled trial of the immunogenicity and tolerability of a meningococcal group C conjugate vaccine in young British infants

A double-blind, randomised, controlled trial was conducted in 248 British infants to assess the immunogenicity and tolerability of three doses of a meningococcal group C/CRM (197) conjugate vaccine (Lederle Laboratories, USA) given at 2, 3 and 4 months. Control children received three doses of Hepatitis B vaccine (Engerix B(R); SmithKline Beecham). At 5 months of age, 100% of children receiving the conjugate vaccine had specific immunoglobulin G concentrations >2.0 microg/ml (n=116) compared with only 4% of control children (n=121). Those receiving the conjugate also had 2.5- and 1.6-fold higher geometric mean concentrations of PRP and diphtheria antibodies, respectively. The vaccine was well tolerated.     

Use of an oral diphtheria vaccine in human

Starch microparticles have been shown to be effective as a particulate adjuvant carrier in oral vaccination. In mice, formulations with bacterial antigens have elicited both systemic and mucosal immune responses providing protection upon challenge with live bacteria. A vaccine formulation with formaldehyde-treated diphtheria toxin cross-reacting material, CRM197, optimised in mice, was tested in healthy volunteers in a booster design. Specific antibodies as well as toxin-neutralising antibodies in a Vero cell analysis indicated that the vaccine was not effective in man. It is obvious that the longer transit time in the human GI tract and possible unfavourable distribution of Peyer's patches and M-cells necessary for the uptake of the starch particles require a more stable formulation. It is proposed that enteric coating of the particles or particles in a gastro-resistant capsule could be a more efficacious vaccine formulation.     

Double-blind study comparing the immunogenicity of a licensed DTwPHib-CRM197 conjugate vaccine (Quattvaxem) with three investigational, liquid formulations using lower doses of Hib-CRM197 conjugate

We performed a double-blind clinical study to evaluate the safety and immunogenicity of four formulations of a DTwPHib full liquid vaccine, three of which contained fractional doses of the 10 microg-dose of CRM197-Hib conjugate vaccine. A total of 261 infants were enrolled and randomised to receive at 3, 4 and 5 months of age, in a double-blind fashion, one of the four DTwPHib vaccine formulations containing 10, 5, 2.5 or 1.25 microg of CRM197-Hib conjugate. Post-immunization reactions were similar in the four vaccine groups, they were mild, transient and resolved without sequelae. The seroconversion rates to anti-PRP titres > or = 0.15 microg/mL were 100%, 98%, 97% and 98% in the groups 10, 5, 2.5 and 1.25 microg, respectively. The seroconversion rates to anti-PRP titres > or =1 microg/mL were 95%, 97%, 88% and 90%, again respectively. Anti-PRP GMTs were 18, 17, 7.82 and 6.94 microg/mL, respectively. All subjects were protected against tetanus and diphtheria, and >80% seroconverted to pertussis. High, and similar, levels of anti-PRP GMTs were elicited by the formulations with 10 and 5 microg of CRM197-Hib conjugate. Although the formulations with 2.5 and 1.25 microg of CRM197-Hib elicited lower levels of anti-PRP GMTs, they were immunogenic and are possible candidates for further development.     

Safety and immunogenicity of a killed Leishmania (L.) amazonensis vaccine against cutaneous leishmaniasis in Colombia: a randomized controlled trial

The safety and immunogenicity of an intramuscular (i.m.) and intradermal (ID) formulation of autoclaved Leishmania (Leishmania) amazonensis vaccine was evaluated in 296 volunteers in a randomized, placebo-controlled, double-blind trial in Colombia. There were 4 vaccination groups: i.m. vaccine, i.m. placebo, ID vaccine, and ID placebo. The ID formulations were mixed with BCG as adjuvant at the time of injection. For each group, 3 vaccinations were given with a 20-day interval between injections, and adverse events were monitored at 20 min, and at 2, 7 and 21 days after each injection. BCG-induced adverse reactions resulted in cancellation of the third vaccine administration in the ID groups. Antibody titres did not differ significantly between the groups. Montenegro skin-test conversion was achieved by 86.4% and 90% of the i.m. vaccine group and by 25% and 5% of the i.m. placebo group 80 days and 1 year after vaccination, respectively. A significant increase in mean Leishmania-antigen lymphocyte proliferation indexes was observed after i.m. vaccine immunization, but not after i.m. placebo immunization, 80 days and 1 year after vaccination. Significant levels of IFN gamma but not IL-10 were observed 1 year after vaccination in the i.m. vaccine group compared to the i.m. placebo group. The good safety profile and evidence of Th1 immune reactions due to i.m. vaccination in this phase-I/II study suggest that a population-based phase-III efficacy trial of the i.m. vaccine should be initiated.     

IgG subclass distribution of antibodies after vaccination of adults with pneumococcal conjugate vaccines

The serum IgG subclass response of adults to Streptococcus pneumoniae (Pnc) capsular polysaccharides (PS) 6B, 14 and 23F was measured for four Pnc vaccines: the 23-valent PS vaccine or PS-protein conjugates with diphtheria toxoid (PncD), tetanus protein (PncT) or CRM197 protein (PncCRM) carriers. A standardized enzyme-linked immunosorbent assay specific for IgG subclasses was employed. This assay uses pneumococcal reference serum, lot 89-SF, to which anti-Pnc PS IgG subclass concentrations have been assigned. Both IgG1 and IgG2 responses were more frequent and higher in the conjugate groups than in the PS group. IgG subclasses in subjects vaccinated with PS displayed similar IgG2 predominant distribution previously observed in both natural and vaccine-induced antibodies. Antibodies induced by PncT, however, had a significantly altered IgG2/IgG1 ratio (P < 0.05), with a higher proportion of IgG1.     

Preclinical evaluation of an investigational 21-valent pneumococcal conjugate vaccine,V116, in adult-rhesus monkey,rabbit, and mouse models

Despite the widespread effectiveness of pneumococcal conjugate vaccines on the overall incidence of invasive pneumococcal disease, the global epidemiological landscape continues to be transformed by residual disease from non-vaccine serotypes, thus highlighting the need for vaccines with expanded disease coverage. To address these needs, we have developed V116, an investigational 21-valent non-adjuvanted pneumococcal conjugate vaccine (PCV), containing pneumococcal polysaccharides (PnPs) 3, 6A, 7F, 8, 9N, 10A, 11A, 12F, 15A, 16F, 17F, 19A, 20, 22F, 23A, 23B, 24F, 31, 33F, 35B, and a de-O-acetylated 15B (deOAc15B) individually conjugated to the nontoxic diphtheria toxoid CRM197 carrier protein. Preclinical studies evaluated the immunogenicity of V116 in adult monkeys, rabbits, and mice. Following one dose, V116 was found to be immunogenic in preclinical animal species and induced functional antibodies for all serotypes included in the vaccine, in addition to cross-reactive functional antibodies to serotypes 6C and 15B. In these preclinical animal studies, the increased valency of V116 did not result in serotype-specific antibody suppression when compared to lower valent vaccines V114 or PCV13. In addition, when compared with naïve controls, splenocytes from V116 to immunized animals demonstrated significant induction of CRM197-specific T cells in both IFN-γ and IL-4 ELISPOT assays, as well as Th1 and Th2 cytokine induction through in vitro stimulation assays, thus suggesting the ability of V116 to engage T cell dependent immune response pathways to aid in development of memory B cells. V116 also demonstrated significant protection in mice from intratracheal challenge with serotype 24F, a novel serotype not contained in any currently licensed vaccine.     

Effect of infant immunisation with meningococcus serogroup C-CRM(197) conjugate vaccine on diphtheria immunity and reactogenicity in pre-school aged children

The majority of Men C conjugate vaccines given in the UK use CRM(197), a mutant diphtheria toxoid, as their protein carrier. We studied the effects of prior immunisation with Men C-CRM(197) conjugate vaccine on immunity to diphtheria in 193 children before and after a booster dose of Men C at 4 years. Baseline diphtheria antibodies were higher in children given four previous doses of Men C (P<0.0001) and tended to be higher following boosting in those who had received three or four doses. This enhanced immunity was not associated with increased reactogenicity.