Preclinical evaluation of the saponin derivative GPI-0100 as an immunostimulating and dose-sparing adjuvant for pandemic influenza vaccines

With the current global influenza vaccine production capacity the large demand for vaccines in case of a pandemic can only be fulfilled when antigen dose sparing strategies are employed. Here we used a murine challenge model to evaluate the potential of GPI-0100, a semi-synthetic saponin derivative, to serve as a dose-sparing adjuvant for influenza subunit vaccine. Balb/c mice were immunized with different doses of A/PR8 (H1N1) subunit antigen alone or in combination with varying doses of GPI-0100. The addition of GPI-0100 significantly stimulated antibody and cellular immune responses, especially of the Th1 phenotype. Furthermore, virus titers detected in the lungs of mice challenged one week after the second immunization were significantly reduced among the animals that received GPI-0100-adjuvanted vaccines. Remarkably, adjuvantation of subunit vaccine with GPI-0100 allowed a 25-fold reduction in hemagglutinin dose without compromising the protective potential of the vaccine.     

Vaccination with HPV16 L2E6E7 fusion protein in GPI-0100 adjuvant elicits protective humoral and cell-mediated immunity

A vaccine comprising human papillomavirus type 16 (HPV16) L2, E6 and E7 in a single tandem fusion protein (termed TA-CIN) has the potential advantages of both broad cross-protection against HPV transmission through induction of L2 antibodies able to cross neutralize different HPV types and of therapy by stimulating T cell responses targeting HPV16 early proteins. However, patients vaccinated with TA-CIN alone develop weak HPV neutralizing antibody and E6/E7-specific T cell responses. Here we test TA-CIN formulated along with the adjuvant GPI-0100, a semi-synthetic quillaja saponin analog that was developed to promote both humoral and cellular immune responses. Subcutaneous administration to mice of TA-CIN (20 μg) with 50 μg GPI-0100, three times at biweekly intervals, elicited high titer HPV16 neutralizing serum antibody, robust neutralizing titers for other HPV16-related types, including HPV31 and HPV58, and neutralized to a lesser extent other genital mucosatropic papillomaviruses like HPV18, HPV45, HPV6 and HPV11. Notably, vaccination with TA-CIN in GPI-0100 protected mice from cutaneous HPV16 challenge as effectively as HPV16 L1 VLP without adjuvant. Formulation of TA-CIN with GPI-0100 enhanced the production of E7-specific, interferon γ producing CD8⁺ T cell precursors by 20-fold. Vaccination with TA-CIN in GPI-0100 also completely prevented tumor growth after challenge with 5 × 10⁴ HPV16-transformed TC-1 tumor cells, whereas vaccination with TA-CIN alone delayed tumor growth. Furthermore, three monthly vaccinations with 125 μg of TA-CIN and 1000 μg GPI-0100 were well tolerated by pigtail macaques and induced both HPV16 E6/E7-specific T cell responses and serum antibodies that neutralized all HPV types tested.     

A murine model for the study of immune memory in response to pneumococcal conjugate vaccination

We developed a murine model for assessment of immunological memory and antibody-induced protection to nasopharyngeal (NP) challenges. BALB/c female mice (n = 10 mice per study parameter) were immunized with two priming doses of the licensed 7-valent pneumococcal (Pnc) conjugate vaccine and immune responses [antibody immunoglobulin G (IgG) levels, avidity and opsonophagocytic activity] were monitored for 26 weeks until IgG levels decreased to nearly baseline. A booster dose of either 2 microg conjugate or 5 microg polysaccharide vaccine was given at week 26. The ability of these two treatments to recall immune memory established by the conjugate vaccine was determined for types 4 and 14 for up to 63 days post-booster. The ability of challenge with pneumococcal type 14 to recall the immune response was also evaluated, as well as, the number of antibody secreting cells (ASC) specific to polysaccharide (Ps) 4, 6B, and 14. A higher dose of conjugate vaccine (2 microg) was necessary to elicit a significant increase in IgG levels after priming with one dose. Priming with lower doses (0.5 and 1.0 microg) only elicited modest increases in IgG levels. Recall of the immune response was found with either conjugate or Ps vaccines. NP challenge with type 14 at week 26 did not recall the immune response, although reduction in NP Pnc load was seen post-primary immunization at 5, 10 and 26 weeks. ASCs were detected in response to either conjugate or Ps booster doses. This model allows for the screening and determination of potential alternative vaccination regimens and the study of immunological markers of memory following Pnc vaccination.     

Studies with inactivated equine influenza vaccine. 1. Serological responses of ponies to graded doses of vaccine.

Serological responses to three bivalent aqueous equine influenza vaccines of different potency and an adjuvanted bivalent vaccine containing inactivated A/equine/Prague/56 (H7N7) and A/equine/Miami/63 (H3N8) viruses, were examined in seronegative ponies. Potencies of the vaccines, measured by single-radial-diffusion tests, ranged from 4 to 56 micrograms of haemagglutinin (HA) antigen activity/virus strain per dose. Serological responses to vaccination were examined by haemagglutination-inhibition (HI) and single-radial-haemolysis (SRH) tests. Four weeks after a primary dose, HI responses to both vaccine viruses were barely detectable; after a second dose the HI responses to A/Miami/63 virus were low or undetectable but HI responses to A/Prague/56 virus were higher (17/20 ponies with titres greater than or equal to 1:16). In contrast SRH tests revealed dose-related antibody responses to both virus strains after one and two vaccine doses; levels after the second dose were 2- to 5-fold higher than after the primary dose. Highest post-vaccination antibody titres were obtained with the adjuvanted vaccine which contained 2- to 4-fold less antigen (13-23 micrograms HA) than the most potent aqueous vaccine. Post-vaccination antibody reacted well in SRH tests with recent antigenic variants of equine influenza virus. A remarkable finding was the high rate of decline in antibody, detected by HI or SRH tests, following one or two doses of vaccine. Even in animals with the highest post-vaccine antibody levels 2-4 weeks after a booster dose, antibody levels had declined to low or indetectable levels 14 weeks later. The low antibody titres detected at 14-32 weeks after vaccination were nevertheless vaccine dose-related.     

Safety and immunogenicity of a recombinant hemagglutinin vaccine for H5 influenza in humans

Recent outbreaks of avian influenza in humans have demonstrated the need for vaccines for influenza viruses with pandemic potential. Recombinant hemagglutinins are an attractive option for such vaccines because they do not require handling potentially highly pathogenic influenza viruses for vaccine production. In order to evaluate the immunogenicity, optimum dosing and timing of administration of a recombinant baculovirus-expressed H5 HA (rH5) in humans, 147 healthy adults were assigned randomly to receive intramuscular rH5 as two doses of 25, 45 or 90 microg each, one dose of 90 microg followed by a dose of 10 microg, or two doses of placebo, at intervals between doses of 21, 28 or 42 days. All doses of rH5 were well tolerated. The rH5 vaccine was modestly immunogenic at high dose. Neutralizing antibody responses to a titer of 1:80 or greater were seen in 23% (14/60) of individuals after a single dose of 90 microg, and in 52% (15/29) after two doses of 90 microg. Varying intervals between doses from 21 to 42 days had no significant effect on antibody responses to vaccination. These results suggest that baculovirus-expressed H5 HA can induce functional antibody in individuals who have not had prior exposure to H5 viruses, but that further studies to improve the immunogenicity of the vaccine are needed.     

Studies with inactivated equine influenza vaccine. 1. Serological responses of ponies to graded doses of vaccine

Serological responses to three bivalent aqueous equine influenza vaccines of different potency and an adjuvanted bivalent vaccine containing inactivated A/equine/Prague/56 (H7N7) and A/equine/Miami/63 (H3N8) viruses, were examined in seronegative ponies. Potencies of the vaccines, measured by single-radial-diffusion tests, ranged from 4 to 56 micrograms of haemagglutinin (HA) antigen activity/virus strain per dose. Serological responses to vaccination were examined by haemagglutination-inhibition (HI) and single-radial-haemolysis (SRH) tests. Four weeks after a primary dose, HI responses to both vaccine viruses were barely detectable; after a second dose the HI responses to A/Miami/63 virus were low or undetectable but HI responses to A/Prague/56 virus were higher (17/20 ponies with titres greater than or equal to 1:16). In contrast SRH tests revealed dose-related antibody responses to both virus strains after one and two vaccine doses; levels after the second dose were 2- to 5-fold higher than after the primary dose. Highest post-vaccination antibody titres were obtained with the adjuvanted vaccine which contained 2- to 4-fold less antigen (13-23 micrograms HA) than the most potent aqueous vaccine. Post-vaccination antibody reacted well in SRH tests with recent antigenic variants of equine influenza virus. A remarkable finding was the high rate of decline in antibody, detected by HI or SRH tests, following one or two doses of vaccine. Even in animals with the highest post-vaccine antibody levels 2-4 weeks after a booster dose, antibody levels had declined to low or indetectable levels 14 weeks later. The low antibody titres detected at 14-32 weeks after vaccination were nevertheless vaccine dose-related.     

Effect of immunization with herpes simplex virus type-1 (HSV-1) glycoprotein D (gD) plus the immune enhancer GPI-0100 on infection with HSV-1 or HSV-2

These studies were performed to determine the effects of GPI-0100, a semi synthetic Quillaja Saponin analog, formulated with herpes simplex virus type-1 (HSV-1) glycoprotein D (gD) on immunity to HSV. SKH-1 hairless mice, used as a model of herpes labialis, inoculated with HSV-1 results in facial lesions, virus replication and mortality. Mortality rates, lesion scores and viral titers were significantly reduced in SKH-1 mice immunized with gD/GPI-0100 prior to cutaneous inoculation with HSV-1 and the protective effects were greater than those using the standard alum adjuvant. Genital HSV-2 infections in guinea pigs were also utilized to determine if gD combined with GPI-0100 was protective against infection, disease severity and viral shedding. Guinea pigs immunized with HSV-1 gD with or without GPI-0100 had significantly reduced area under the curve lesion scores, but infection rates and virus shedding was not altered. When Tween 40 was added to gD and GPI-0100, mean peak lesion scores were also significantly reduced. The results obtained in a genital HSV-2 infection of guinea pigs did not indicate enhanced protection or reduced virus shedding following immunization with GPI-0100 and gD. There was, however, a significant improvement in clinical herpetic genital disease with the combination of gD plus the immune enhancer GPI-0100.     

Phase I and II randomised trials of the safety and immunogenicity of a prototype adjuvanted inactivated split-virus influenza A (H5N1) vaccine in healthy adults

OBJECTIVE: The primary objective was to evaluate the safety and immunogenicity of a prototype inactivated, split-virus H5N1 (avian influenza A) vaccine. A secondary objective was to assess the cross-reactivity of immune responses to two variant clade 2 H5N1 strains. METHODS: In two randomised, dose comparison, parallel assignment, multicentre trials conducted in Australia, healthy adult volunteers received two doses of 7.5 microg or 15 microg H5 haemagglutinin (HA) vaccine+/-AlPO4 adjuvant (phase I trial; N=400) or two doses of 30 microg or 45 microg H5 HA with AlPO4 adjuvant (phase II trial; N=400). Revaccination with a booster dose was offered 6 months after dose 2 (phase I trial only). Main outcome measures were the change in immunogenicity at each follow-up visit from baseline, measured using HA inhibition (HI) and virus microneutralisation (MN) assays, and the frequency and nature of adverse events (AEs). Computer generated tables were used to randomly allocate treatments; participants and investigators were blinded to treatment allocation. FINDINGS: All formulations were well-tolerated; no unexpected serious adverse events were reported. Two doses of 30 microg or 45 microg H5 HA adjuvanted formulations elicited the highest immune responses, with considerable MN antibody (>or=1:20) persistence up to 6 months post-vaccination. The 7.5 and 15 microg formulations (+/-adjuvant) were less immunogenic than the higher dose formulations; HI and MN antibody titres decreased to near pre-vaccination levels at 6 months but were restored to post-dose 2 levels after the booster dose. Immune responses in the phase I trial demonstrated modest levels of cross-protective MN antibodies against two currently circulating, distinct clade 2 H5N1 strains. INTERPRETATION: Two doses of prototype 30 microg or 45 microg aluminium-adjuvanted, clade 1 H5N1 vaccines were immunogenic and well-tolerated with considerable 6-month antibody persistence. The prototype H5N1 vaccine also elicited modest levels of cross-protective MN antibodies against variant clade 2 H5N1 strains [ClinicalTrials.gov identifiers: NCT00136331, NCT00320346; Funding: CSL Limited, Australia].     

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.     

Safety and immunogenicity of a new Neisseria meningitidis serogroup C-tetanus toxoid conjugate vaccine in healthy adults.

We evaluated the safety and immunogenicity of a single dose of a new serogroup C O-deacetylated meningococcal polysaccharide-tetanus toxoid conjugate vaccine in 30 healthy adult volunteers. The vaccine was well tolerated with no serious adverse events and minimal local reactions and systemic symptoms. All subjects developed a fourfold or greater increase in serum bactericidal antibody (SBA) to serogroup C meningococcus. SBA geometric mean titre increased from 11 to 3649 (p<0.001). Serogroup C-specific IgG levels increased postvaccination from 0.65 to 17.02 microg/ml (p<0.001). Bactericidal titres pre- and postimmunisation showed significant correlation with serogroup C-specific IgG (r(2)=0.693). Antibody levels fell by 6 months postvaccination, however, meningococcal C IgG avidity increased indicating the successful induction of a T-cell-dependent antibody response. Conclusion: meningococcal C-tetanus toxoid conjugate vaccine is immunogenic and well tolerated in healthy adults.     

河豚毒素实验疫苗的不同剂量对免疫效应的影响

目的　为提高河豚毒素抗毒实验疫苗的效价 ,探索不同免疫剂量对免疫反应的影响。方法　以河豚毒素 (TTX)与中国鲎血蓝蛋白 (TTH)的化学偶联物TTX TTH免疫Balb/c小鼠 ,比较高低不同免疫剂量 (分别为每次 10 0和 2 5 μg)对抗体质量和疫苗的抗毒效价的影响。以血清抗体质量和TTX攻毒效果检验疫苗的效价。结果　低剂量组的抗体质量升速比高剂量组快。两者相比 ,单次抗TTX中毒效价 :半数活存剂量分别为 16×LD和 11×LD ,最大耐受剂量分别为 2 6×LD和 2 2×LD ;累积抗毒效价 :半数动物分别可累计耐受 4 0×LD和 2 2×LD以上 ,测得最大累计耐受剂量分别为 10 4×LD和 90×LD。结论　TTX的实验疫苗在不同免疫剂量方案均有效预防了TTX的攻毒。低剂量组比高剂量组有更好的抗体质量、单次抗毒效价和累积抗毒效价。     

Effect of individual conjugate dose on immunogenicity of type 6B pneumococcal polysaccharide-N. meningitidis outer membrane protein complex conjugate vaccines in infant rhesus monkeys

A pneumococcal conjugate vaccine (PCV) has been developed consisting of capsular polysaccharide (Ps) coupled to the outer membrane protein complex of Neiserria meningitidis serogroup B. Experiments were conducted in infant rhesus monkeys to assess the potential to administer multiple Pn types in a single vaccine. A single type conjugate, 6B, was dosed from 0.025 to 25 microg Ps. Peak anti-6B Ps Ab titers were seen at lower doses of 0.025 and 0.25 microg Ps, while reduced titers of anti-6B Ps Ab were observed at the highest doses of conjugate administered, 2.5 and 25 microg Ps. By mixing free Ps, carrier, or another monovalent PCV with this 6B PCV, it was determined that reduced anti-6B Ps titers at high PCV doses were associated only with the quantity of type-specific Ps in the conjugate. Thus, increasing the amount of carrier protein or adding an additional monovalent conjugate did not significantly affect the response to type 6B Ps. These results suggest that, given an appropriately determined dose per individual pneumococcal Ps type, a multivalent PCV that includes many different types should have satisfactory clinical immunogenicity.     

Evaluation of meningococcal serogroup C conjugate vaccine programs in Canadian children: interim analysis

Background

To assess antibody titers afforded by meningococcal C- (MenC) tetanus toxoid conjugate vaccine at 12 months of age in three different immunization schedules.

Methods

This prospective study included three similar cohorts of healthy infants from 1-dose, 2-dose and 3-dose MenC infant immunization programs. Infants were enrolled at 12 months of age and given the final scheduled dose of MenC-tetanus toxoid conjugate vaccine with sera collected prior to and 1 month after the vaccination. Serum bactericidal activity (SBA) titers ≥ 1:8 were considered protective.

Results

Before the 12 month dose, participants had significantly different protective titers according to the number of prior doses received: 100% (95% CI 97.6–100%) of infants who had 2 prior doses (at 2 and 4 months) were protected compared to 84.0% (76.7–89.3%) of participants with one dose (at 2 months) and 27.6% (21.0–35.4%) of unvaccinated infants. All subjects were protected after the 12 month MenC dose, but titers were higher with prior priming.

Conclusions

Two MenC doses given in infancy afford optimal protection during the first year of life; however, substantial protection was seen after one dose at 2 months.     

Immunogenicity and safety of a tetravalent dengue vaccine and a bivalent HPV vaccine given concomitantly or sequentially in girls aged 9 to 14 years in Mexico

Dengue is endemic in several regions, and the global incidence is increasing. The recombinant, live, attenuated, tetravalent dengue vaccine (CYD-TDV) is recommended for dengue seropositive individuals ≥ 9 years. Human papillomavirus (HPV) vaccination is recommended for girls aged 9–14 years to prevent HPV infection-related cancers. This study assessed the immunogenicity and safety of a bivalent HPV (types 16 and 18) vaccine and CYD-TDV when co-administered concomitantly or sequentially.This was a Phase IIIb, randomized, open-label, multicenter study in girls aged 9–14 years in Mexico (NCT02979535). Participants were randomized 1:1 to receive three doses of CYD-TDV 6 months apart and two doses of bivalent HPV vaccine either concomitantly with, or 1 month before (sequentially), the first 2 CYD-TDV doses. Antibody levels were measured at baseline and 28-days after each vaccine dose for all participants, using an enzyme-linked immunosorbent assay for HPV-16 and HPV-18 antibodies, and a plaque reduction neutralization test for the four dengue serotypes; results are reported only for participants who were seropositive at baseline. Safety was assessed for all randomized participants throughout the study.Of the randomized participants, 305/478 (63.8%) were seropositive for dengue at baseline: 154 in the concomitant group and 151 in the sequential group. After the last HPV vaccine dose, the antibody titers for HPV were comparable in seropositive participants between treatment groups, with between group titer ratios of 0.966 for HPV-16 and 0.999 for HPV-18. After dose 3 of CYD-TDV, antibody titers were comparable for the concomitant and sequential groups across all serotypes, with between-group ratios close to 1 (serotype 1: 0.977; serotype 2: 0.911; serotype 3: 0.921; serotype 4: 0.931).CYD-TDV and a bivalent HPV vaccine administered concomitantly or sequentially in dengue seropositive girls aged 9–14 years elicited comparable immune responses with similar safety profiles.     

Low dose revaccination induces robust protective anti-HBs antibody response in the majority of healthy non-responder neonates

A sizeable proportion (1-10%) of healthy adults and to lesser extent neonates vaccinated with triple 10 microg hepatitis B (HB) vaccine fail to mount a protective antibody response. Revaccination with the same vaccine dose has proved to be effective in a significant number of primary non-responders. The influence of revaccination with lower vaccine doses however has not been studied adequately in non-responder neonates. This study was conducted to evaluate the influence of supplementary vaccination with a single low and standard dose of a recombinant hepatitis B (HB) vaccine in healthy Iranian non-responder neonates to primary vaccination. Iranian neonates unable to respond to primary vaccination with 10, 5 or 2.5 microg doses of recombinant HB vaccine were revaccinated with a single additional dose of the same concentration. Serum anti-HBs antibody titer was measured by sandwich ELISA. Administration of a single additional dose induced seroprotection (anti-HBs> or =10IU/L) in 10/12 (83%), 10/12 (83%) and 21/24 (87.5%) of non-responder neonates in 10, 5 and 2.5 microg vaccine recipients with geometric mean titers (and 95% confidence limits) of 1358 (258-7142), 401 (79-2038) and 164 (62-433) IU/L, respectively. The log-transformed antibody titer obtained for the 10 microg dose recipients was significantly higher than that of the 2.5 microg dose vaccinees (p=0.028). No significant differences in anti-HBs titer were observed between other groups of vaccinees. However, the total seroprotection rates obtained after administration of four low doses of 2.5 and 5 microg were significantly higher than that obtained after administration of the classical three 10 microg doses (p=0.029 and p=0.006, respectively). The total seroprotection rates were similar between all groups of vaccines receiving four doses of 2.5, 5 and 10 microg vaccine doses. These results indicate that a significant proportion of non-responder neonates can be induced to develop a protective anti-HBs response following revaccination with a single low dose vaccine. Thus adaptation of four low dose (2.5 or 5 microg) vaccination is expected to induce higher seroprotection rate and lower or comparable anti-HBs antibody titer in healthy neonates.     

Therapeutic efficacy of a human papillomavirus type 16 E7 bacterial exotoxin fusion protein adjuvanted with CpG or GPI-0100 in a preclinical mouse model for HPV-associated disease

Persistent human papillomavirus (HPV) infection is causally linked to the development of several human cancers, including cervical, vulvar, vaginal, anal, penile, and oropharyngeal cancers. To address the need for a therapeutic vaccine against HPV-associated diseases, here we test and compare the immunogenicity and therapeutic efficacy of a bacterial exotoxin fusion protein covalently linked to the HPV16 E7 oncoprotein adjuvanted with CpG or GPI-0100 in the C3.43 preclinical HPV16-transformed tumor model. We show that TVGV-1 protein vaccine adjuvanted with either CpG or GPI-0100 adjuvant induces a high frequency of E7-specific CD8⁺ T cells, and both adjuvants are able to assist the immune response in inducing polyfunctional cytokine-secreting lytic T cells that show therapeutic efficacy against well-established C3.43 tumors. CpG-adjuvanted TVGV-1 resulted in higher frequencies of IFNγ secreting and degranulating E7-specific T cells compared to GPI-0100-adjuvanted TVGV-1, resulting in marginally increased in vivo efficacy. Despite minor differences in immune response outcomes, we consider both CpG ODN and GPI-0100 to be promising vaccine adjuvants to increase the immunogenicity and therapeutic efficacy of the TVGV-1 protein for HPV16-driven cancers.     

Effectiveness and safety of mutant Escherichia coli heat-labile enterotoxin (LT H44A) as an adjuvant for nasal influenza vaccine

The effectiveness and safety of mutant Escherichia coli heat-labile enterotoxin, LT H44A (His to Arg substitution at position 44 from the N-terminus of the A1 fragment of the A subunit) as an adjuvant for nasal influenza vaccine were examined. (1) When 0.2 microg of LT H44A, together with 0.2 microg of influenza A/PR/8/34 virus (PR8, H1N1) vaccine, was administered intranasally into BALB/c mice (twice, 4 weeks apart), anti-PR8 hemagglutinin (HA) IgA and IgG antibody (Ab) responses were induced at levels that were sufficient to provide either complete protection against infection with a small volume of PR8 virus suspension or partial protection against infection with a lethal dose of the suspension. The dose of the mutant LT and vaccine used here (0.2 microg/ 20 g doses mouse) corresponded to the estimated dose per person, i.e. 0.1 mg/10 kg body weight. (2) Using these vaccination conditions, no additional total IgE Ab responses were induced. (3) The mutant was confirmed to be less toxic than the native LT when the toxicity was analyzed either using Y1 adrenal cells in vitro (1/483 EC(50)) or by an ileal loop test. (4) One hundred micrograms of the mutant, administered intranasally or intraperitoneally into guinea-pigs (Heartley strain, 0.3-0.4 kg), caused no body-weight changes 7 days after administration, although 100 microg of the native LT administered intraperitoneally caused death in all guinea-pigs due to diarrhea within 2 days. The intranasal administration of 100 microg of the mutant resulted in almost no pathological changes in the nasal mucosa 3 days after administration. These results suggest that LT H44A, which can be produced in high yields in an E. coli culture (about 5 mg/l), could be used as one of the effective and safe adjuvants for nasal influenza vaccine in humans.     

A bivalent Neisseria meningitidis recombinant lipidated factor H binding protein vaccine in young adults: Results of a randomised, controlled, dose-escalation phase 1 trial

Neisseria meningitidis is a leading cause of meningitis and septicaemia, but a broadly-protective vaccine against endemic serogroup B disease is not licensed and available. The conserved, outer-membrane lipoprotein factor H binding protein (fHBP, also known as LP2086) is expressed as one of two subfamily variants in virtually all meningococci. This study investigated the safety, tolerability, and immunogenicity of a recombinant-expressed bivalent fHBP (r-fHBP) vaccine in healthy adults. Participants (N=103) aged 18-25 years were recruited into three ascending dose level cohorts of 20, 60, and 200μg of a bivalent r-fHBP vaccine formulation and randomised to receive vaccine or placebo at 0, 1, and 6 months. The vaccine was well tolerated. Geometric mean titres (GMTs) for r-fHBP subfamily-specific IgG antibodies increased 19-168-fold from pre-vaccination to post-dose 2 in a dose level-dependent manner. In addition, robust serum bactericidal assay using human complement (hSBA) responses for strains expressing both homologous and heterologous fHBP variants were observed. After three vaccinations, 16-52% of the placebo group and 47-90%, 75-100%, and 88-100%, of the 20, 60, and 200μg dose levels, respectively, had seroprotective (≥1:4) hSBA titres against six serogroup B strains. The bivalent r-fHBP vaccine was well tolerated and induced robust bactericidal activity against six diverse serogroup B strains in young adults at the 60 and 200μg dose levels.     

Co-administration of morphine and oxycodone vaccines reduces the distribution of 6-monoacetylmorphine and oxycodone to brain in rats

Opioid conjugate vaccines have shown promise in animal models as a potential treatment for opioid addiction. Individual vaccines are quite specific and each targets only a limited number of structurally similar opioids. Since opioid users can switch or transition between opioids, we studied a bivalent immunization strategy of combining 2 vaccines that could target several of the most commonly abused opioids; heroin, oxycodone and their active metabolites. Morphine (M) and oxycodone (OXY) haptens were conjugated to keyhole limpet hemocyanin (KLH) through tetraglycine (Gly)(4) linkers at the C6 position. Immunization of rats with M-KLH alone produced high titers of antibodies directed against heroin, 6-monoacetylmorphine (6-MAM) and morphine. Immunization with OXY-KLH produced high titers of antibodies against oxycodone and oxymorphone. Immunization with the bivalent vaccine produced consistently high antibody titers against both immunogens. Bivalent vaccine antibody titers against the individual immunogens were higher than with the monovalent vaccines alone owing, at least in part, to cross-reactivity of the antibodies. Administration of a single concurrent intravenous dose of 6-MAM and oxycodone to rats immunized with the bivalent vaccine increased 6-MAM, morphine and oxycodone retention in serum and reduced the distribution of 6-MAM and oxycodone to brain. Vaccine efficacy correlated with serum antibody titers for both monovalent vaccines, alone or in combination. Efficacy of the individual vaccines was not compromised by their combined use. Consistent with the enhanced titers in the bivalent group, a trend toward enhanced pharmacokinetic efficacy with the bivalent vaccine was observed. These data support the possibility of co-administering two or more opioid vaccines concurrently to target multiple abusable opioids without compromising the immunogenicity or efficacy of the individual components.     

Human IgG subclass responses in relation to serum bactericidal and opsonic activities after immunization with three doses of the Norwegian serogroup B meningococcal outer membrane vesicle vaccine

Ten adult volunteers, with low prevaccination levels of serum IgG antibodies against meningococcal antigens (< 1 microg ml(-1)), received three doses of the Norwegian group B meningococcal outer membrane vesicle (OMV) vaccine intramuscularly at weeks 0, 6 and 46. Anti-OMV IgG subclass responses were measured and compared with serum bactericidal activity (SBA) and opsonic activity against the vaccine strain 44/76. All vaccinees showed an IgG1 antibody response after each vaccine dose. The vaccine-induced median serum IgG1 antibody levels were 16, 17 and 18 microg ml(-1) 2-6 weeks after the first, second and third dose, respectively. Three vaccinees showed a weak IgG3 response after the first dose, whereas 8 and 9 showed a response after the second (median = 10 microg ml(-1)) and third dose (median = 10 microg ml(-1)), respectively. Low levels of anti-OMV IgG2 antibodies were found, whilst specific IgG4 antibodies were only detected for one vaccinee. The vaccine induced at least a fourfold increase in SBA titre in 8 vaccinees after the first dose, in 9 vaccinees after 2 doses and in all vaccinees after 3 doses. A positive correlation was found between IgG1 subclass antibody levels and SBA (r = 0.62, P < 0.0001). Elevated opsonophagocytic activity, measured as respiratory burst (RB), was observed in all vaccinees after one vaccine dose and usually increased after 2 and 3 doses. A strong positive correlation was found between IgG1 antibody levels and RB (r = 0.76, P < 0.0001). In conclusion, we have shown that systemic meningococcal OMV vaccination in adult vaccinees mainly induced IgG1 antibodies which correlated with bactericidal and opsonic activity, but also a considerable amount of IgG3 antibodies, which, in contrast to the IgG1 response, was induced only after 2 or 3 vaccine doses and declined more rapidly.