Search

Background: We evaluated immunogenicity of SCB-2019, a subunit vaccine candidate containing a pre-fusion trimeric form of the SARS-CoV-2 spike (S)-protein adjuvanted with CpG-1018/alum., Methods: The phase 2/3, double-blind, randomized SPECTRA trial was conducted in five countries in participants aged ≥ 18 years, either SARS-CoV-2-naïve or previously exposed. Participants were randomly assigned to receive two doses of SCB-2019 or placebo administered intramuscularly 21 days apart. In the phase 2 part of the study, on days 1, 22, and 36, neutralizing antibodies were measured by pseudovirus and wild-type virus neutralization assays to SARS-CoV-2 prototype and variants, and ACE2-receptor-binding antibodies and SCB-2019-binding antibodies were measured by ELISA. Cell-mediated immunity was measured by intracellular cytokine staining via flow cytometry., Results: 1601 individuals were enrolled between 24 March and 13 September 2021 and received at least one vaccine dose. Immunogenicity analysis was conducted in a phase 2 subset of 691 participants, including 428 SARS-CoV-2-naïve (381 vaccine and 47 placebo recipients) and 263 SARS-CoV-2-exposed (235 vaccine and 28 placebo recipients). In SARS-CoV-2-naïve participants, GMTs of neutralizing antibodies against prototype virus increased 2 weeks post-second dose (day 36) compared to baseline (224 vs 12.7 IU/mL). Seroconversion rate was 82.5 %. In SARS-CoV-2-exposed participants, one SCB-2019 dose increased GMT of neutralizing antibodies by 48.3-fold (1276.1 IU/mL on day 22) compared to baseline. Seroconversion rate was 92.4 %. Increase was marginal post-second dose. SCB-2019 also showed cross-neutralization capability against nine variants, including Omicron, in SARS-CoV-2-exposed participants at day 36. SCB-2019 stimulated Th1-biased cell-mediated immunity to the S-protein in both naïve and exposed participants. The vaccine was well tolerated, no safety concerns were raised from the study., Conclusions: A single dose of SCB-2019 was immunogenic in SARS-CoV-2-exposed individuals, whereas two doses were required to induce immune response in SARS-CoV-2-naïve individuals. SCB-2019 elicited a cross-neutralizing response against emergent SARS-CoV-2 variants at antibody levels associated with clinical protection, underlining its potential as a booster., Clinicaltrials: gov: NCT04672395; EudraCT: 2020-004272-17., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: MH received financial support from Clover Biopharmaceuticals, grants from the European Union, the Belgian Health Care Knowledge Centre, and Université Libre de Bruxelles, travel fees from Gilead and Pfizer, financial support from INSMED, is involved in the Belgian Society of Infectious Diseases and Clinical Microbiology, the working group on Belgian COVID-19 therapeutic guidelines, and the Belgian Task Force on therapeutics for COVID-19. GS served as a Scientific Advisory Board Member and received consulting fees from Clover Biopharmaceuticals, AdVaccine, CanSino, Everest Medicines, Valneva, Vaxart, Affinivax, Genocea, and Vaxxess. DA received consulting fees from Clover Biopharmaceuticals, Vaxxinity, Everest Medicines, Senda, served as a Scientific Advisory Board Member for Clover Biopharmaceuticals, Vaxxinity, Senda, Everest Medicines, and Inventprise, and served as a board member for Clover Biopharmaceuticals and Inventprise. RC received funding from the Bill & Melinda Gates Foundation, consulting fees from Icosavax, Hillevax, honoraria from AstraZeneca, served as a board member for Clover Biopharmaceuticals, Curevac, IVI, Inventprise, and owns stocks in Icosavax, Hillevax, Curevac, Novartis, Roche, GSK, and Clover Biopharmaceuticals. EB had contracts with Clover Biopharmaceuticals, Astra-Zeneca, Janssen, GSK, Merck, and Moderna. ERA, JCC, LFBF, and MEBM declare that they have no competing financial interests. BH, HHH, HQ, and HLC are employees of Clover Biopharmaceuticals. CB is an employee of Clover Biopharmaceuticals owns stocks in Clover Biopharmaceuticals, GSK, and Sanofi. PL and IS are employees of Clover Biopharmaceuticals and own stocks in the company., (Copyright © 2023. Published by Elsevier Ltd.)

Background: Pneumococcal diseases remain prevalent despite available polysaccharide and conjugate vaccines. This phase 1/2 study evaluated safety/tolerability and immunogenicity of a novel 24-valent pneumococcal vaccine (ASP3772) based on high-affinity complexing of proteins and polysaccharides., Methods: Pneumococcal vaccine-naïve adults aged 18-85 years were randomized to receive either ASP3772 or PCV13 (13-valent conjugate vaccine). Participants received a single intramuscular injection of ASP3772 (1-, 2-, or 5-µg dose per polysaccharide) or PCV13. A separate, nonrandomized group of PCV13-vaccinated participants (65-85 years) received PPSV23 (23-valent polysaccharide vaccine). Assessments were obtained through Day 7 for reactogenicity, through Day 30 for safety and tolerability, and through Month 6 for serious adverse events. Immunogenicity was measured at Day 30 using assays for functional opsonophagocytic activity (OPA) and pneumococcal serotype-specific anticapsular polysaccharide immunoglobulin G for each serotype., Results: In both age cohorts, the most frequently reported local reactions were self-limited tenderness and pain after ASP3772 at all dose levels or after PCV13, occurring within 2-3 days. Fatigue, headache, and myalgia were the most frequently reported systemic reactions following either vaccine. Robust OPA responses for all serotypes were observed across all ASP3772 dose groups in both age cohorts. Older adults (aged 65-85 years) who received ASP3772 had significantly higher immune responses to several PCV13 serotypes and all non-PCV13 serotypes than participants who received PCV13. OPA responses to the ASP3772 5-µg dose were significantly higher for several serotypes in naïve participants than in older adults with prior exposure to PCV13 who were administered PPSV23 in this study., Conclusions: These results demonstrate that ASP3772 is well tolerated, highly immunogenic, and in adults may offer significantly broader protection than existing pneumococcal vaccines., Clinicaltrials: gov: NCT03803202., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: [Gurunadh R. Chichili reports financial support was provided by Astellas Pharma Global Development Inc. Ronald Smulders reports financial support was provided by Astellas Pharma Global Development Inc. Vicki Santos reports financial support was provided by Astellas Pharma Global Development Inc. Beth Cywin reports financial support was provided by Astellas Pharma Global Development Inc. Laura Kovanda reports financial support was provided by Astellas Pharma Global Development Inc. Charles Van Sant reports financial support was provided by Astellas Pharma Global Development Inc. Frank Malinoski reports a relationship with Affinivax that includes: employment. Shite Sebastian reports a relationship with Affinivax that includes: employment. George Siber reports a relationship with Affinivax that includes: consulting or advisory and employment. Richard Malley reports a relationship with Affinivax that includes: consulting or advisory and employment. Richard Malley has issued and pending patents on MAPS vaccines, Rhizavidin-fusion proteins, and Multivalent Pneumococcal Vaccine. Shite Sebastian has issued and pending patents on Multivalent Pneumococcal Vaccine., (Copyright © 2022 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Background: We previously reported the efficacy of the adjuvanted-protein COVID-19 vaccine candidate S-Trimer (SCB-2019) in adults who showed no evidence of previous exposure to SARS-CoV-2. In this study, we aimed to investigate the extent of protection afforded by previous exposure to SARS-CoV-2 on subsequent COVID-19 infection, as well as the efficacy, safety, and reactogenicity of SCB-2019 in participants who were enrolled in the Study evaluating Protective-Efficacy and safety of Clover's Trimeric Recombinant protein-based and Adjuvanted COVID-19 vaccine (SPECTRA) trial who had already been exposed to SARS-CoV-2 before vaccination., Methods: In a phase 2 and 3 multicentre, double-blind, randomised, placebo-controlled trial (SPECTRA) done at 31 sites in five countries, participants were randomly assigned 1:1 using the Cenduit Interactive Response Technology system (IQVIA, Durham, NC, USA), with a block size of six, to receive two doses of either SCB-2019 or placebo 21 days apart. The primary outcomes of the SPECTRA trial were vaccine efficacy, measured by real-time PCR (rtPCR)-confirmed COVID-19 of any severity, with onset from 14 days after the second vaccine dose, as well as the safety and solicited local and systemic adverse events in the phase 2 subset. Here, we present secondary analyses to calculate the protective efficacy due to previous exposure to SARS-CoV-2 against reinfection with COVID-19 according to severity in SPECTRA participants who had evidence of exposure to SARS-CoV-2 at baseline, including efficacy against identified viral variants, as well as efficacy of SCB-2019 vaccination in this population., Findings: We enrolled 30 174 participants between March 24, 2021, and Aug 10, 2021. In the 14 670 participants who were randomly assigned to receive placebo, there were 418 (2·8%) confirmed cases of COVID-19; 65 (0·9%) of 7339 SARS-CoV-2-exposed participants, and 353 (4·8%) of 7331 SARS-CoV-2-naive participants (attack rates of 5·5 cases per 100 person-years for SARS-CoV-2-exposed participants and 32·4 cases per 100 person-years for SARS-CoV-2-naive participants). Protective efficacy due to previous exposure to SARS-CoV-2 was 83·2% (95% CI 78·0-87·3) against any COVID-19, 92·5% (82·9-97·3) against moderate-to-severe COVID-19, and 100% (59·3-100) against severe COVID-19; no SARS-CoV-2-exposed participants had hospitalisation associated with COVID-19. Protective efficacy against variants were 100% for alpha (B.1.1.7) and lambda (C.37) variants, 88·6% (14·9-99·7) for B.1.623, 93·6% (80·1-98·7) for gamma (P.1), and 92·4% (81·2-97·6) for mu (B.1.621) variants, and lowest against beta (B.1.351; 72·2% [33·1-89·9]) and delta (B.1.617.2; 77·2% [61·3-87·2]) variants. In addition, one dose of SCB-2019 had 49·9% (1·5-75·6) efficacy against any symptomatic COVID-19, and two doses had 64·2% (26·5-83·8) efficacy. SCB-2019 was well tolerated in SARS-CoV-2-exposed participants, but was associated with higher rates of injection site pain (89 [33·8%] of 263 participants) than placebo (16 [6·7%] of 239 participants). Rates of solicited systemic adverse events, severe adverse events, and serious adverse events were similar between vaccine and placebo groups, and with rates in SARS-CoV-2-naive vaccine recipients., Interpretation: Previous exposure to SARS-CoV-2 decreased the risk and severity of subsequent COVID-19 infection, even against newly emerging variants. Protection is further enhanced by one or two doses of SCB-2019., Funding: Clover Biopharmaceuticals, The Coalition for Epidemic Preparedness Innovations (CEPI)., Competing Interests: Declaration of interests IS, HHH, PL, CB, CV, and JL are full-time employees of Clover Biopharmaceuticals. FR is a statistical adviser, and SACC, RC, DA, PR, and GS are scientific advisers for Clover Biopharmaceuticals., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Background: A range of safe and effective vaccines against SARS CoV 2 are needed to address the COVID 19 pandemic. We aimed to assess the safety and efficacy of the COVID-19 vaccine SCB-2019., Methods: This ongoing phase 2 and 3 double-blind, placebo-controlled trial was done in adults aged 18 years and older who were in good health or with a stable chronic health condition, at 31 sites in five countries (Belgium, Brazil, Colombia, Philippines, and South Africa). The participants were randomly assigned 1:1 using a centralised internet randomisation system to receive two 0·5 mL intramuscular doses of SCB-2019 (30 μg, adjuvanted with 1·50 mg CpG-1018 and 0·75 mg alum) or placebo (0·9% sodium chloride for injection supplied in 10 mL ampoules) 21 days apart. All study staff and participants were masked, but vaccine administrators were not. Primary endpoints were vaccine efficacy, measured by RT-PCR-confirmed COVID-19 of any severity with onset from 14 days after the second dose in baseline SARS-CoV-2 seronegative participants (the per-protocol population), and the safety and solicited local and systemic adverse events in the phase 2 subset. This study is registered on EudraCT (2020-004272-17) and ClinicalTrials.gov (NCT04672395)., Findings: 30 174 participants were enrolled from March 24, 2021, until the cutoff date of Aug 10, 2021, of whom 30 128 received their first assigned vaccine (n=15 064) or a placebo injection (n=15 064). The per-protocol population consisted of 12 355 baseline SARS-CoV-2-naive participants (6251 vaccinees and 6104 placebo recipients). Most exclusions (13 389 [44·4%]) were because of seropositivity at baseline. There were 207 confirmed per-protocol cases of COVID-19 at 14 days after the second dose, 52 vaccinees versus 155 placebo recipients, and an overall vaccine efficacy against any severity COVID-19 of 67·2% (95·72% CI 54·3-76·8), 83·7% (97·86% CI 55·9-95·4) against moderate-to-severe COVID-19, and 100% (97·86% CI 25·3-100·0) against severe COVID-19. All COVID-19 cases were due to virus variants; vaccine efficacy against any severity COVID-19 due to the three predominant variants was 78·7% (95% CI 57·3-90·4) for delta, 91·8% (44·9-99·8) for gamma, and 58·6% (13·3-81·5) for mu. No safety issues emerged in the follow-up period for the efficacy analysis (median of 82 days [IQR 63-103]). The vaccine elicited higher rates of mainly mild-to-moderate injection site pain than the placebo after the first (35·7% [287 of 803] vs 10·3% [81 of 786]) and second (26·9% [189 of 702] vs 7·4% [52 of 699]) doses, but the rates of other solicited local and systemic adverse events were similar between the groups., Interpretation: Two doses of SCB-2019 vaccine plus CpG and alum provides notable protection against the entire severity spectrum of COVID-19 caused by circulating SAR-CoV-2 viruses, including the predominating delta variant., Funding: Clover Biopharmaceuticals and the Coalition for Epidemic Preparedness Innovations., Competing Interests: Declaration of interests IS, HHH, PLi, RH, CB, BH, and JL are full-time employees of Clover Biopharmaceuticals. FR is a statistical adviser for Clover Biopharmaceuticals. RC, DA, PR, and GS are scientific advisers for Clover Biopharmaceuticals. All other authors declare no competing interests., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Correlates of protection for COVID-19 vaccines are urgently needed to license additional vaccines. We measured immune responses to four COVID-19 vaccines of proven efficacy using a single serological platform. IgG anti-Spike antibodies were highly correlated with ID50 neutralization in a validated pseudoviral assay and correlated significantly with efficacies for protection against infection with wild-type, alpha and delta variant SARS-CoV-2 virus. The protective threshold for each vaccine was calculated for IgG anti-Spike antibody. The mean protective threshold for all vaccine studies for WT virus was 154 BAU/ml (95 %CI 42-559), and for studies with antibody distributions that enabled precise estimation of thresholds (i.e. leaving out 2-dose mRNA regimens) was 60 BAU/ml (95 %CI 35-102). We propose that the proportion of individuals with responses above the appropriate protective threshold together with the geometric mean concentration can be used in comparative non-inferiority studies with licensed vaccines to ensure that new vaccines will be efficacious., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: [Dr. Plotkin consults for Janssen and Moderna; Dr. Siber reports personal fees and other from Clover Biopharmaceuticals, personal fees from AdVaccine, other from Vaxxinity personal fees from CanSino, personal fees from CureVac, personal fees from Valneva, personal fees from Vaxart, personal fees and other from Affinivax, outside the submitted work; Dr. Ambrosino reports personal fees from Vaxxinity, personal fees and other from Clover Biopharmaceuticals, outside the submitted work. Dr. Montefiori’s laboratory receives funding from Moderna for clinical sample testing]., (Copyright © 2021 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

A significant correlation has been shown between the binding antibody responses against original severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein and vaccine efficacy of 4 approved coronavirus disease 2019 vaccines. We therefore assessed the immune response against original SARS-CoV-2 elicited by the adjuvanted S-Trimer vaccine, SCB-2019 + CpG/alum, in the same assay and laboratory. Responses to SCB-2019 were comparable or superior for antibody to original and Alpha variant when compared with 4 approved vaccines. The comparison accurately predicted success of the recently reported efficacy trial of SCB-2019 vaccine. Immunogenicity comparisons to original strain and variants of concern should be considered as a basis for authorization of vaccines., (© The Author(s) 2021. Published by Oxford University Press for the Infectious Diseases Society of America.)

Pulmonary surfactant (PS) is a surface active complex of lipids and proteins that prevents the alveolar structures from collapsing and reduces the work of breathing by lowering the surface tension at the alveolar air-liquid interface (ALI). Surfactant is synthesized by the alveolar type II (AT II) cells, and it is stored in specialized organelles, the lamellar bodies (LBs), as tightly packed lipid bilayers. Upon secretion into the alveolar lining fluid, a large fraction of these particles retain most of their packed lamellar structure, giving rise to the term lamellar body like-particles (LBPs). Due to their stability in aqueous media, freshly secreted LBPs can be harvested from AT II cell preparations. However, when LBPs get in contact with an ALI, they quickly and spontaneously adsorb into a highly organized surface film. In the present study we investigated the adsorptive capacity of LBPs at an ALI under relevant physiological parameters that characterize the alveolar environment in homeostatic or in pathological conditions. Adsorption of LBPs at an ALI is highly sensitive to pH, temperature and albumin concentration and to a relatively lesser extent to changes in osmolarity or Ca(2+) concentrations in the physiological range. Furthermore, proteolysis of LBPs significantly decreases their adsorptive capacity confirming the important role of surfactant proteins in the formation of surface active films., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Healthy adults aged ≥ 70 years (N=443) with no history of pneumococcal vaccination received 7- or 9-valent pneumococcal conjugate vaccine (PCV7 or PCV9) at 1 × (PCV7 only), 2 × (PCV7+PCV9), or 4 × (2 × PCV7+2 × PCV9) dosage in a randomised, open-label study evaluating pneumococcal protein conjugate vaccine (PnC). Controls received 23-valent pneumococcal polysaccharide vaccine (PPV). Both geometric mean concentration enzyme-linked immunosorbent assay and opsonophagocytic activity antibody titres assessed 1 month after vaccination were significantly increased over baseline titres for all PCV7 serotypes, with a trend toward a dose-dependent immune response. Local reactions for the 4 × dose, but not the 2 × dose, were statistically significantly higher than for the 1 × dose. No treatment-related serious adverse events occurred., (Copyright © 2011. Published by Elsevier Ltd.)

The term "sense of coherence" (SOC) refers to a life perspective which spans affective states centering around notions of comprehensibility, manageability, meaningfulness of work or personal contribution, and self-confidence, in a context where demands are perceived as challenges. SOC is positively correlated with psychological well-being, stress management and negatively with anxiety, depression, and neuroticism. General practitioners licensed by the regional health insurance fund in Styria were the subject of a 29-item survey on Sense of Coherence as described by Antonovsky; the return rate was 78.3%. The SOC of GPs replying to the survey was categorized as "marginal" in 29.9% of cases, "significant" in 59.9%, and "rigid" (in the sense of a so-called "inauthentic" self-image) in 10.2% of cases. Overall, the value found was 5.30 +/- 0.56 (Likert scale = absolute 153.8 +/- 16.2; significant), which falls within the range of values for other comparable services or professions, and comes slightly on top over figures for a normative sample. No significant correlations were found for either age or gender (p > 0.05). Reported values for comprehensibility gained in proportion to the age of the general practice of the informant (p < 0.05).

Background: Pneumococcal conjugate vaccines are important for the prevention of serious illness and death among infants. Factors associated with pneumococcal conjugate vaccine immunogenicity have not been explored., Methods: Children <24 months of age received 2, 3, or 4 doses of 7-valent pneumococcal conjugate vaccine (PnCRM7) or control vaccine depending on age at enrollment. Serum samples were tested for serotype-specific antibodies by enzyme-linked immunosorbant assay. Multiple linear regression was used to determine predictors of immunogenicity., Results: Among 315 PnCRM7-vaccinated subjects and 295 control subjects enrolled at <7 months of age, geometric mean concentrations (GMCs) of antibodies were significantly higher after dose 3 than after dose 2 for all serotypes except type 4. The proportion of subjects with antibody concentrations > or =5.0 micro g/mL was higher for all serotypes, but the proportion with concentrations > or =0.35 micro g/mL was higher only for types 6B and 23F. Three-dose and 2-dose regimens for those 7-11 and 12-23 months of age, respectively, were highly immunogenic. Increased maternal antibody concentrations were associated with reduced responses to dose 1 and 3 but not to dose 4 of PnCRM7., Conclusions: Maternal antibody is associated with a reduced infant response to PnCRM7 but does not interfere with immune memory. In infants, a third priming dose increases the antibody GMC and the proportion achieving an antibody concentration > or =5.0 micro g/mL but has little impact on the proportion achieving a concentration > or =0.35 micro g/mL.

The World Health Organization (WHO) is undertaking a series of consultations on serological criteria for the evaluation and licensure of new formulations/combinations or different vaccination schedules of pneumococcal conjugate vaccines. The lack of a definitive serological correlate of protection and the multiplicity of antigens involved, especially since the clinical efficacy of most of the individual serotypes represented in the only licensed vaccine has not been established, are hindering the formulation of criteria for licensure of new formulations or combinations of the vaccine. This report analyses the various options with their relative merits and drawbacks and provides preliminary recommendations as guidance to regulatory agencies in evaluating these vaccines for the purposes of licensure. More detailed recommendations for production and control of pneumococcal conjugate vaccines, including criteria for evaluation for licensure, are currently being drafted.

Background: Immunization of healthy women before pregnancy is a potential approach to providing increased levels of maternal antibody to newborns to protect them from infections occurring during the perinatal period and first months of life., Methods: Healthy nonpregnant Pima Indian women of childbearing age were randomized to receive one of two Haemophilus influenzae type b (Hib) conjugate vaccines [HbOC or Hib-meningococcal outer membrane protein complex (OMP)] or a 23-valent pneumococcal polysaccharide vaccine (PnPs). Infants received Hib-OMP vaccine at 2, 4 and 12 months of age. Vaccine safety and immunogenicity was evaluated in the women and their infants., Results: Anti-polyribose ribitol phosphate antibody titers were significantly higher in women in both Hib conjugate vaccine groups than in the pneumococcal vaccine group throughout the 37-month observation period. Antibody responses to HbOC vaccine were significantly higher than those to Hib-OMP. A subsequent booster dose of each Hib conjugate vaccine induced reactions and antibody responses similar to those of the first dose. Infants born to mothers immunized with Hib vaccines compared with PnPs had significantly higher polyribose ribitol phosphate-specific IgG antibody titers at birth and 2 months of age but lower antibody responses to Hib-OMP at 6 months and similar titers before and after boosting with Hib-OMP at 1 year of age. By contrast women immunized with PnPs did not have significantly elevated concentrations of pneumococcal-specific antibody at delivery, and their infants had pneumococcal antibody titers similar to those of infants born to mothers who did not receive pneumococcal vaccine before pregnancy., Conclusion: Hib conjugate vaccine given to women before pregnancy significantly increased the proportion of infants who had protective Hib antibody levels at birth and 2 months of age.

The development of glycoconjugate vaccines for Streptococcus pneumoniae that are effective in very young children has renewed interest in identification of which among the more than 90 pneumococcal serotypes are most likely to cause invasive pneumococcal disease (IPD). Serotype distribution is thought to vary geographically, even between regions as socioeconomically similar as western Europe and North America. To explain these variations, we note the considerable variation that exists between reported rates of IPD in young children in the USA and west European countries. We postulate that this variation is attributable to different blood-culture rates and practices, and that mild IPD is probably underdiagnosed and under-reported in western Europe. On the basis of a comparison of serotype distributions between the two regions, we also postulate that those serotypes found at similar frequencies in both regions are virulent and rarely cause mild disease. As a result, reported distributions of IPD serotypes, especially when expressed as percentages, might be strongly skewed by the distribution of clinical presentations in a particular study population.

Objective: To determine the efficacy, safety and immunogenicity of the heptavalent CRM197 pneumococcal conjugate vaccine against invasive disease caused by vaccine serotypes and to determine the effectiveness of this vaccine against clinical episodes of otitis media., Methods: The Wyeth Lederle Heptavalent CRM197 (PCV) was given to infants at 2, 4, 6 and 12 to 15 months of age in a double blind trial; 37,868 children were randomly assigned 1:1 to receive either the pneumococcal conjugate vaccine or meningococcus type C CRM197 conjugate. The primary study outcome was invasive disease caused by vaccine serotype. Other outcomes included overall impact on invasive disease regardless of serotype, effectiveness against clinical otitis media visits and episodes, impact against frequent and severe otitis media and ventilatory tube placement. In addition the serotype-specific efficacy against otitis media was estimated in an analysis of spontaneously draining ears., Results: In the interim analysis in August, 1998, 17 of the 17 cases of invasive disease caused by vaccine serotype in fully vaccinated children and 5 of 5 of partially vaccinated cases occurred in the control group for a vaccine efficacy of 100%. Blinded case ascertainment was continued until April, 1999. As of that time 40 fully vaccinated cases of invasive disease caused by vaccine serotype had been identified, all but 1 in controls for an efficacy of 97.4% (95% confidence interval, 82.7 to 99.9%), and 52 cases, all but 3 in controls in the intent-to-treat analysis for an efficacy of 93.9% (95% confidence interval, 79.6 to 98.5%). There was no evidence of any increase of disease caused by nonvaccine serotypes. Efficacy for otitis media against visits, episodes, frequent otitis and ventilatory tube placement was 8.9, 7.0, 9.3 and 20.1% with P < 0.04 for all. In the analysis of spontaneously draining ears, serotype-specific effectiveness was 66.7%., Conclusion: This heptavalent pneumococcal conjugate appears to be highly effective in preventing invasive disease in young children and to have a significant impact on otitis media.

Objectives: The objectives of this study were (1) to determine the safety and immunogenicity of heptavalent pneumococcal CRM197 conjugate (PNCRM7) vaccine in infants and (2) to determine the effect of concurrent hepatitis B immunization during the primary series and the effect of concurrent diphtheria and tetanus toxoid and acellular pertussis [DTaP (ACEL-IMUNE)] and conjugate CRM197 Haemophilus influenzae type b [HbOC (HibTITER) immunization at time of the booster dose on the safety and immunogenicity of PNCRM7and these other concurrently administered vaccines., Methods: This was a randomized double-blinded study in 302 healthy infants in the Northern California Kaiser Permanente (NCKP) Health Plan. Infants received either PNCRM7 vaccine or meningococcal group C conjugate vaccine as a control at 2, 4 and 6 months of age and a booster at 12 to 15 months of age. Study design permitted the evaluation of immunology and safety of concurrent administration of routine vaccines. Antibody titers were determined on blood samples drawn before and 1 month after the primary series and the booster dose., Results: After the third dose of PNCRM7 geometric mean concentrations (GMCs) ranged from 1.01 for serotype 9V to 3.72 microg/ml for serotype 14. More than 90% of all subjects had a post-third dose titer of > or =0.15 microg/ml for all serotypes, and the percentage of infants with a post-third dose titer of > or =1.0 microg/ml ranged from 51% for type 9V to 89% for type 14. After the PNCRM7 booster dose, the GMCs of all seven serotypes increased significantly over both post-Dose 3 and pre-Dose 4 antibody levels. In the primary series there were no significant differences in GMCs of pneumococcal antibodies between the subjects given PN-CRM7 alone or concurrently with hepatitis B vaccine. At the toddler dose concurrent administration of PNCRM7 and DTaP and HbOC resulted in a near conventional threshold for statistical significance of a post-Dose 4 GMC for serotype 23F [alone 6.75 mirog/ml vs. concurrent 4.11 microg/ml (P = 0.057)] as well as significantly lower antibody GMCs for H. influenza polyribosylribitol phosphate, diphtheria toxoid, pertussis toxin and filamentous hemagglutinin. For all antigens there were no differences between study groups in defined antibody titers that are considered protective., Conclusion: We conclude that PNCRM7 vaccine was safe and immunogenic. When this vaccine was administered concurrently at the booster dose with DTaP and HbOC vaccines, lower antibody titers were noted for some of the antigens when compared with the antibody response when PNCRM7 was given separately. Because the GMCs of the booster responses were all generally high and all subjects achieved similar percentages above predefined antibody titers, these differences are probably not clinically significant.

Pertussis in infants is often severe, resulting in prolonged hospitalization. Treatment is limited to supportive care. Antibiotics do not significantly alter the course of the disease unless administered during the catarrhal phase. Therapies directed at pertussis toxin, a major virulence factor of Bordetella pertussis, may be beneficial. This study uses the aerosol challenge model to further examine the protective effects of P-IGIV, a new intravenous immunoglobulin product, which has high levels of pertussis toxin antibodies. P-IGIV was prepared as a 4% immunoglobulin G (IgG) solution from the pooled donor plasma from donors immunized with inactivated pertussis toxoid. The IgG pertussis toxin antibody concentration in P-IGIV is >7-fold higher than conventional intravenous immunoglobulin products. In the aerosol challenge model, P-IGIV-treated mice exhibited a dose-dependent decrease in mortality when monitored for 28 days postchallenge. P-IGIV in doses of 2,800, 1,400, and 350 mg/kg significantly reduced mortality compared to saline (P < 0.01)- and human IGIV (P < 0.01)-treated controls. The 50% protective dose of pertussis toxin antibodies in P-IGIV was 147 microg/ml. Recovery of weight gain and normalization of leukocyte counts occurred in all P-IGIV-treated groups but did not exhibit dose-dependent characteristics. Even after 7 days of infection, P-IGIV reversed the effects of pertussis in mice. This study provides further evidence that pertussis toxin antibodies not only play a role in passive protection but can also reverse symptoms of established disease in mice. We feel that P-IGIV deserves further evaluation in children hospitalized with severe pertussis.

Background: Pertussis in infants is often severe, resulting in complications and prolonged hospitalization. Treatment is limited to supportive care. Antibiotics do not significantly alter the course of the disease. Therapies directed at pertussis toxin, a major virulence factor of Bordetella pertussis, might be beneficial. This study examines the safety and pharmacology of intravenous pertussis immunoglobulin (P-IGIV), which has high levels of pertussis toxin antibodies., Methods: P-IGIV was prepared as a 4% IgG solution from the pooled plasma from donors immunized with inactivated pertussis toxoid. The IgG pertussis toxin antibody concentration of 733 microg/ml is >7-fold higher than contained in conventional intravenous immunoglobulin products. Children with presumptive pertussis were allocated to one of three treatment doses of P-IGIV., Results: Twenty-six of 30 enrolled children had confirmed pertussis. There were no adverse events associated with P-IGIV except one patient who had transient hypotension that responded to an infusion rate decrease. P-IGIV doses of 1500, 750 and 250 mg/kg achieved > or =4-fold, 3-fold and >2-fold rises in peak geometric mean titers of pertussis toxin IgG antibodies, respectively. P-IGIV exhibited a half-life of 38.4 days and a volume of distribution of 87.8 ml/kg. All three treatment groups showed declines in lymphocytosis (P < 0.05) and paroxysmal coughing by the third day after P-IGIV infusion compared with preinfusion values., Conclusion: P-IGIV is safe and achieves high pertussis toxin antibody titers in infants. This study provides data for a prospective, controlled trial of P-IGIV.

A guinea pig model to assess the immunogenicity of a combination vaccine containing diphtheria, tetanus and acellular pertussis (DTaP) vaccine and Haemophilus influenzae type b (Hib) capsular polysaccharide conjugated to tetanus toxoid (HibT) was evaluated comparatively with the mouse immunogenicity test to study the effect of combining these antigens on the immunogenicity of various components. The immunogenicity test in mice was performed by subcutaneous injection of groups of 10 animals twice at an interval of four weeks with 1/10 of a single human dose of various formulations of combination vaccines, DTaP or HibT vaccine. The animals were bled at 4 and 6 weeks and IgG or total antibodies to various components were determined by ELISA or RIA. The guinea pig immunogenicity model included groups of animals injected subcutaneously twice at an interval of six weeks with 1.5 times the single human dose of various formulations. The animals were bled at 4, 6 and 8 weeks and serum samples were tested for antibodies to various components by ELISA, RIA and/or neutralization tests. Additionally, potency of tetanus and diphtheria components was assessed as per the US Food and Drug Administration's regulations. Aluminium phosphate (AIPO(4)) adsorbed HibT vaccine or HibT as a combination with AIPO(4)adsorbed DTaP vaccine showed significant increases in IgG antibodies to tetanus toxin in mice as well increased tetanus antitoxin levels in guinea pigs as compared to soluble HibT vaccine. In general, combining DTaP and HibT vaccines did not affect the antibody levels to tetanus and diphtheria toxoids whereas DTaP-HibT combination vaccine elicited significantly lower IgG antibodies to pertussis toxin and filamentous haemagglutinin than DTaP vaccine alone, particularly after first injection. Mice showed similar Hib antibody responses for the combination and HibT alone whereas guinea pigs consistently showed lower anamnestic responses to Hib for combination formulations than for HibT alone. Reducing the amount of HibT and/or tetanus toxoid in the combination formulations reduced this suppression of Hib antibody response in guinea pigs. Suppression of Hib antibody response in combination vaccines has also been reported from recent clinical trials. Based on the results from this study, it appears that the guinea pig model may be able to predict the human response to various components of combination vaccines., (Copyright 1999 The International Association for Biologicals.)

Asplenic patients are at increased risk for life-threatening infections with polysaccharide-encapsulated organisms, and reports of responses to polysaccharide vaccines have been conflicting. Thirty-six asplenic patients and 15 healthy controls were immunized with pneumococcal, Haemophilus influenzae type b (Hib), and meningococcal vaccines. Antibody concentrations to Hib and pneumococcal serotypes 14 and 18C were measured by ELISA. IgG antibody responses to all three antigens were similar in asplenic patients and controls at 28 days following immunization. In contrast, asplenic patients had significantly lower IgM concentrations in response to Hib (P<.05) and to both pneumococcal serotypes 14 (P<. 005) and 18C (P<.001). IgA anti-Hib antibody was also lower in the asplenic group, as was total anti-Hib antibody measured by RIA. These results document that IgG responses to polysaccharide vaccines are normal in asplenic patients. The impaired IgM responses of these patients may explain conflicting reports from studies that measured only total antibody-binding concentrations.

The authors developed a simple and rapid method for quantitation of free capsular polysaccharide of Haemophilus influenzae type b (polyribosyl ribitol phosphate, PRP) in PRP-tetanus toxoid conjugate vaccine based on acid precipitation of tetanus toxoid (TT). Acid hydrolysis of PRP during the assay was not detected. The conditions used in the assay did not precipitate unconjugated PRP or adipic acid dihydrazide derivatized PRP. The method was highly reliable, reproducible and sensitive. The accuracy of the assay was confirmed by spiking known amounts of unconjugated PRP to PRP-TT conjugate preparations. A PRP-TT preparation, incubated at 37 degrees C for 6 months showing most of the PRP as unconjugated (87% determined by this method), was not immunogenic in mice for the PRP component even after two injections. In contrast, the same preparation held at 4 degrees C for 20 months, showing 17% unconjugated PRP, induced IgG antibodies to PRP which were boosted after second injection. Therefore, this method is very useful to evaluate the stability of PRP-TT conjugate vaccine. The assay may be useful for characterizing other polysaccharide-protein conjugate vaccines.

Polymyxin B (PMB) is a cyclic decapeptide antibiotic which also binds and neutralizes endotoxin. Unfortunately, PMB can be considerably nephrotoxic at clinically utilized doses, thereby limiting its utility as a therapeutic antiendotoxin reagent. We sought to change the pharmacokinetics and toxicity profile of PMB by covalently linking it to a human immunoglobulin G (IgG) carrier. Conjugates of PMB with IgG were prepared by EDAC [1-ethyl-3-(3-dimethylaminopropyl) carbodiimide]-mediated amide formation. Analysis by dot enzyme-linked immunosorbent assay with an anti-PMB monoclonal antibody showed that the purified conjugate contained bound PMB. The IgG-PMB conjugate reacted with lipid A and J5 lipopolysaccharide in Western blot assays in a manner comparable to that of whole antiserum with anti-lipid A reactivity; unconjugated IgG had no reactivity. The PMB bound in the conjugate retained its endotoxin-neutralizing activity compared to that of unbound PMB as evidenced by its dose-dependent inhibition of tumor necrosis factor release by endotoxin-stimulated human monocytes in vitro; unconjugated IgG had no activity. By this assay, the PMB-IgG conjugate was determined to have approximately 3.0 microg of bound functional PMB per 100 microg of total protein of conjugate (five molecules of PMB per IgG molecule). The PMB-IgG conjugate was also bactericidal against clinical strains of Escherichia coli, Pseudomonas aeruginosa, and Klebsiella pneumoniae relative to unconjugated IgG with MBCs of <4 microg of conjugate per ml for each of the tested strains. The conjugate appeared to be nontoxic at the highest doses deliverable and provided statistically significant protection from death to galactosamine-sensitized, lipopolysaccharide-challenged mice in a dose-dependent fashion when administered prophylactically 2 h before challenge. However, neither free PMB nor the PMB-IgG conjugate could protect mice challenged with endotoxin 2 h after administration. This suggests that these reagents can play a role in prophylaxis but not in therapy of sepsis. These experiments demonstrated that the PMB-IgG conjugate retains bound yet functional PMB as evidenced by its endotoxin-neutralizing activity both in vitro and in vivo. Further work is required to define the role that this or related conjugate compounds may play in the prophylaxis of endotoxin-mediated disease.

We have determined threshold doses of aluminium phosphate (AlPO4) adsorbed tetanus toxoid (TT) and diphtheria toxoid (DT) in mice and guinea pigs with a view to developing an animal model to assess the immunogenicity of controlled release vaccines. A dose was sought (threshold dose) which produces little antibody after primary injection and a moderate response after a booster injection, thus mimicking the adult human response to TT and DT vaccines adsorbed on to aluminium adjuvants. After the first injection, mice and guinea pigs showed a dose response for both tetanus toxin IgG antibodies and tetanus antitoxin over a wide range of doses of AlPO4-adsorbed TT (0.01 to 0.2 Lf). After the second and third injections, there was no clear dose response for doses between 0.05 and 0.2 Lf However, doses between 0.01 and 0.04 Lf still showed a dose response after the second injection. Dilution of AlPO4-adsorbed TT in saline just before injection did not alter immunogenicity after the first injection, but stronger booster responses were seen in mice to diluted versus undiluted vaccine after the second and third injections. The threshold dose of AlPO4-adsorbed TT for both mice and guinea pigs was 0.01 Lf given in 100 microliters. For AlPO4-adsorbed DT, three strains of mice (inbred, Balb/c and C57/B6, and outbred, CD-1) showed a dose response after the first injection for DT IgG antibodies and diphtheria antitoxin at 0.1 and 0.2 Lf doses. Outbred CD-1 mice showed a dose response after the second and third injections also, whereas inbred mice showed inconsistent dose responses after the second injection and none after the third injection. In contrast to AlPO4-adsorbed TT, mice injected with undiluted AlPO4-adsorbed DT elicited significantly higher antibodies than those injected with diluted formulations, particularly after the first injection. The threshold dose of AlPO4-adsorbed DT for mice was 0.1 Lf in a volume of 250 microliters. Lower doses did not produce consistent antibody responses in mice. We propose that a single dose of controlled release formulations at doses not greater than 1/10 of a single human dose when injected into mice and guinea pigs should produce an antibody response similar or higher than two to three threshold doses of AlPO4-adsorbed TT or DT.

Though vaccination has been the most cost-effective way of controlling infectious diseases, the logistics of delivering at least two to three doses of conventional vaccines for primary immunization to achieve protection are difficult and compliance is frequently inadequate, particularly in developing countries. In recent years biodegradable polymer microspheres have received much attention for the purposes of controlled release of antigens, (i) to reduce the number of doses needed for primary immunization to as few as a single dose and (ii) to target an antigen to microfold cells on mucosal surfaces after oral administration or to antigen-presenting cells after parenteral inoculations. A variety of vaccine antigens have been encapsulated in microspheres usually composed of poly (lactic/glycolic) acid (PLGA). Based on the size of the microspheres, molecular weight of polymer and ratio of lactic to glycolic acid in the polymer, the antigen may be targeted to various cells of the immune system or it may form a depot at the site of injection, allowing the slow release of the antigen for extended periods. Additionally, another adjuvant may be incorporated inside microspheres together with the antigen, further enhancing or modulating the immune response to the desired type. The major problems in developing controlled-release vaccines include instability of vaccine antigens during micro-encapsulation, storage and subsequent hydration. We encapsulated tetanus toxoid (TT) and Haemophilus influenzae type b capsular polysaccharide conjugated to TT (Hib-T) inside PLGA microspheres and evaluated the antibody levels in mice. A single injection of these micro-encapsulated vaccines elicited high antibody levels which persisted for several months. The antibody levels were similar or superior to those elicited by conventional formulations of AIPO4-adsorbed TT or soluble Hib-T conjugate vaccine.

The adjuvanticity of the phosphazene polymer, poly[di(carboxylatophenoxy) phosphazene] (PCPP) was examined with a diverse collection of immunogens. PCPP proved to be a potent adjuvant for trivalent influenza virus vaccine, tetanus toxoid, hepatitis B surface antigen, herpes simplex virus glycoprotein gD2 and the capsular polysaccharide, polyribosylribitolphosphate, from Haemophilus influenzae type b. Taken together these results clearly demonstrate the general utility of PCPP as an adjuvant. Furthermore, PCPP was a superior adjuvant at least with TT compared to similar negatively charged polyanions, polymethylacrylic acid and polyacrylic acid.

Objective: To study the kinetics of Haemophilus influenzae type b (Hib)-specific antibody in infants born to mothers immunized with an Hib polysaccharide or one of two Hib conjugate vaccines., Study Design: Serum antibody to the polyribosylribitol (PRP) moiety of Hib was measured by radioimmunoassay and enzyme-linked immunosorbent assay at birth and at 2 and 6 months of age in infants born to women immunized with Hib polysaccharide or conjugate vaccine (PRP-D and HbOC). A subset of infants > or = 6 months of age was immunized with Hib conjugate vaccine after licensure of this vaccine for infants. A comparison group of 18 infants born to unimmunized women received the same Hib conjugate vaccine on a similar schedule., Results: Total PRP antibody concentrations were 1.50, 14.4 and 20.4 microg/ml in 2-month-old infants born to mothers immunized with polysaccharide, PRP-D and HbOC vaccines, respectively, and 2.54, 1.35 and 2.46 microg/ml in 6-month-old infants. Infants born to mothers immunized with polysaccharide vaccine had significantly less PRP antibody at 2 months of age but similar antibody concentrations at 6 months of age. Persistence or increases in total PRP antibody during 6 months were noted in 21 of 47 (44.6%) study infants. A subset of study and comparison infants was immunized with a mean of 2.6 doses of Hib vaccines between 6 months and 2 years of age, and all infants had total PRP antibody concentrations > or = 0.15 microg/ml., Conclusion: Conjugate Hib vaccines administered during the last trimester of pregnancy resulted in significantly higher PRP antibody titers in infants at birth and 2 months of age than did polysaccharide vaccine. A subset of infants born to immunized mothers was subsequently immunized with Hib conjugate vaccine and had antibody concentrations similar to those in infants born to nonimmunized women.

Immunogenicity of tetanus toxoid (TT) encapsulated in biodegradable polymer microspheres composed of poly lactide (PLA) or poly lactide-co-glycolide (PLGA) polymers was evaluated in mice and guinea pigs for 1 year. Microsphere formulations made from polymers differing in molecular weight and composition elicited significantly higher IgG antibody levels than soluble TT in mice. The antibody levels elicited by microsphere formulations in mice and guinea pigs were similar to those elicited by a single injection of AlPO4 adsorbed TT. Immunogenicity was not consistently better with a particular polymer composition, molecular weight or microsphere size. However, animals primed with TT-containing microspheres showed significantly higher anamnestic response to a low dose booster 1 year after priming than those primed with AlPO4 adsorbed TT. Microspheres made from low molecular weight PLGA polymer showed a minimal local tissue reaction 1 year after injection. In contrast, aluminum adjuvant formed local granulomas which persisted for 1 year after injection. Microsphere formulations used in this study released a small fraction of antigenic TT during in vitro release studies due to denaturation of TT during encapsulation and hydration of microspheres. Nevertheless, strong priming of immune responses were seen. It remains to be demonstrated whether stabilization of TT would lead to more immunogenic microsphere formulations.

Immunogenicity of native and formalinized cross reacting material (CRM197) of diphtheria toxin (DTx) was assessed in mice and guinea pigs. For the primary response, mice produced similar levels of diphtheria toxoid (DTxd) IgG antibodies to both native and formalinized preparations of CRM197 though the antibody levels were significantly lower than those elicited by conventional DTxd (P < 0.05). In contrast, guinea pigs showed significantly higher levels of DTxd IgG antibodies to the formalinized CRM197 preparation than the native preparation (P < 0.001) after single injection. These differences in the immunogenicity of mice and guinea pigs to native and formalinized CRM197 preparations have implications for the development and control of diphtheria or other vaccines involving the use of CRMs.

Objective: To determine whether long-term supplementation with vitamin E enhances in vivo, clinically relevant measures of cell-mediated immunity in healthy elderly subjects., Design: Randomized, double-blind, placebo-controlled intervention study., Setting and Participants: A total of 88 free-living, healthy subjects at least 65 years of age., Intervention: Subjects were randomly assigned to a placebo group or to groups consuming 60, 200, or 800 mg/d of vitamin E for 235 days., Main Outcome Measures: Delayed-type hypersensitivity skin response (DTH); antibody response to hepatitis B, tetanus and diphtheria, and pneumococcal vaccines; and autoantibodies to DNA and thyroglobulin were assessed before and after supplementation., Results: Supplementation with vitamin E for 4 months improved certain clinically relevant indexes of cell-mediated immunity in healthy elderly. Subjects consuming 200 mg/d of vitamin E had a 65% increase in DTH and a 6-fold increase in antibody titer to hepatitis B compared with placebo (17% and 3-fold, respectively), 60-mg/d (41% and 3-fold, respectively), and 800-mg/d (49% and 2.5-fold, respectively) groups. The 200-mg/d group also had a significant increase in antibody titer to tetanus vaccine. Subjects in the upper tertile of serum alpha-tocopherol (vitamin E) concentration (>48.4 micromol/L [2.08 mg/dL]) after supplementation had higher antibody response to hepatitis B and DTH. Vitamin E supplementation had no effect on antibody titer to diphtheria and did not affect immunoglobulin levels or levels of T and B cells. No significant effect of vitamin E supplementation on autoantibody levels was observed., Conclusions: Our results indicate that a level of vitamin E greater than currently recommended enhances certain clinically relevant in vivo indexes of T-cell-mediated function in healthy elderly persons. No adverse effects were observed with vitamin E supplementation.

Various methods to determine loading of vaccine in biodegradable polymer microspheres encapsulating tetanus toxoid were evaluated. The microspheres were composed of poly (D-lactic acid) (PLA) and poly (DL-lactic-co-glycolic acid) (PLGA). Dissolution of microspheres in organic solvents such as methylene chloride, chloroform, or dimethyl sulfoxide and extraction of vaccine antigen or total protein with phosphate buffered saline gave variable results which depended upon the characteristics of the microspheres, such as type of polymer, excipients used in the microspheres and formulation conditions. Microspheres made from low molecular weight PLGA polymer and showing a large burst release exhibited up to 25% extraction of antigen whereas microspheres made from PLA microspheres with low burst release showed < 1% extraction. Extraction of total protein with 0.1 N NaOH and 5% sodium dodecyl sulfate showed results similar to those obtained with organic solvent extraction method. Partial digestion of microspheres with 6 N HCl at 60 degrees C for 20 h resulted in approximately 30% loss in TT protein by micro-bicinchoninic acid (BCA) assay. The major problem with this method was strong reactions in the micro-BCA assay of stabilizers, particularly sugars (glucose, sucrose) used in the microsphere formulations. Complete digestion of microspheres with 6 N HCl at 110 degrees C for 20 h or with 13.5 N NaOH at 121 degrees C for 1 h and quantitation of amino acids by a modified ninhydrin assay showed reproducible results on the protein loading in the microspheres. However, this method was affected by the presence of stabilizers, such as gelatin, which contain amino acids. Further, sucrose concentrations higher than 10% caused interference in the ninhydrin assay on samples hydrolyzed with 6 N HCl. In contrast, hydrolysis with 13.5 N NaOH did not show any interference by sucrose. Stabilizers used outside the microspheres for lyophilization purposes may be removed by washing the microspheres before loading determination or by dialysis but stabilizers used inside the microspheres would still cause interference. For reliable determination of total protein in the microspheres containing vaccines, we suggest complete digestion of microspheres with acid or base followed by amino acid analysis by colorimeteric assays such as ninhydrin method or using amino acid analyzers. The method needs to be optimized for each type of formulation to eliminate interference by the excipients. Alternatively, total protein nitrogen in the microspheres may be determined by the Kjel-dahl method if no amino acids or other nitrogen containing stabilizer is used inside the microspheres.

Objective: To compare a recombinant bactericidal/permeability-increasing protein variant and a recombinant endotoxin-neutralizing protein., Design: Randomized, blinded, controlled study, using a rat model of sepsis., Setting: Animal research facility., Subjects: Male Wistar rats., Interventions: An inoculum of 1.5 x 10(7) to 1.8 x 10(8) Escherichia coli O18ac K1, implanted in the peritoneum, produced bacteremia in 95% of animals after 1 hr. One hour after E. coli challenge, animals received recombinant bactericidal/permeability-increasing protein variant, recombinant endotoxin-neutralizing protein, or saline intravenously, followed by ceftriaxone and gentamicin intramuscularly., Measurements and Main Results: Twenty-four (85.7%) of 28 animals receiving recombinant endotoxin-neutralizing protein (p < .001 vs. control) survived 7 days compared with nine (33.3%) of 27 recombinant bactericidal/permeability-increasing protein variant-treated (p < .001 vs. control) and two (6.5%) of 31 control animals., Conclusions: Both recombinant endotoxin-neutralizing protein and recombinant bactericidal/permeability-increasing protein variant improved survival. Recombinant endotoxin-neutralizing protein was superior to recombinant bactericidal/permeability-increasing protein variant in its protective effect at the doses tested. Our results suggest that both proteins may be useful in the treatment of human Gram-negative sepsis.

A high-titered and stable respiratory syncytial virus (RSV) is essential for the development of RSV vaccines and quality control of vaccines and RSV immune globulin. We increased the virus titer of RSV seed stock, and virus preparations made from this seed stock, 100 times by removing defective interfering particles using limiting dilution procedure. RSV preparations made from the new seed stock had infectivity titers ranging from 10(7.6) to 10(8.2) TCID50 per ml for five lots made over a period of 3 years. Unstabilized RSV lost most of its infectivity at -86 degrees C within 2-3 weeks, at 37 degrees C within 24 hr, at 56 degrees C within 3 min and after five freeze-thaw cycles. The high titered virus was stabilized at -86 degrees C for 3 years, at 37 degrees C for 3 days, at 56 degrees C for 6 min and against five freeze-thaw cycles. Most effective stabilizers included 25% sucrose, 10% trehalose and 45% fetal bovine serum (FBS) in Medium 199 whereas 3.5% dimethyl sulfoxide, > or = 45% FBS in phosphate buffered saline, 40% glycerol and 10% sorbitol also stabilized RSV to lesser and variable degrees. A mixture of 0.5% gelatine and 0.3% sodium glutamate stabilized the virus for a short period whereas 0.1 M MgCl2 and 25% FBS did not stabilize the virus. The stabilized high-titered virus is very useful for achieving reproducibility in serologic assays. A broad spectrum of stabilizers, such as those evaluated in this study, would be useful in choosing the most suitable formulation for stabilizing a live RSV vaccine.

Radiolabeled tetanus toxoid (TT) was prepared by detoxifying chromatographically purified tetanus toxin with 14C-labeled formaldehyde. 14C-TT was encapsulated inside poly (D,L-lactide-co-glycolide, 50/50) microspheres (MS) of varying average size (approximately 10 microns and approximately 50 microns). Balb/c mice were injected subcutaneously with 5 Lf (approximately 15 micrograms) of 14C-TT, encapsulated in MS, mixed with blank MS without encapsulated antigen, as soluble antigen or adsorbed onto aluminum phosphate (AlPO4) and radioactivity was monitored at the site of injection, draining lymph nodes, blood, liver, spleen, and kidneys at various intervals. At one day, approximately 95% and 90% radioactivity disappeared from site of injection for soluble TT or blank MS mixed TT and AlPO4 adsorbed TT, respectively, whereas approximately 55% and 70% radioactivity disappeared from site of injection for MS of average size approximately 50 microns and approximately 10 microns, respectively. By 7 days, 99% of radioactivity disappeared from site of injection for soluble TT or blank MS mixed TT, whereas 2-3% radioactivity persisted at the site of injection for AlPO4 adsorbed TT for 4 weeks. In contrast, approximately 20% radioactivity stayed at the site of injection for MS injected mice up to 4 weeks. At all time points, large MS (approximately 50 microns) showed more radioactivity at the site of injection than small MS (approximately 10 microns). Other organs showing radioactivity were draining lymph nodes and kidneys. Small MS with encapsulated TT showed highest level of radioactivity in lymph nodes at 4 h. In kidneys, soluble and AlPO4 adsorbed TT showed a peak of radioactivity at 4 h whereas TT encapsulated in MS showed a peak of radioactivity at 7 days. These results indicate that AlPO4 did not act as a depot for TT at the site of injection, but TT encapsulated in MS did form a depot for approximately 1 month.

Objective: To test the efficacy of a recombinant endotoxin neutralizing protein as compared with saline in rats with Escherichia coli sepsis., Design: Prospective, controlled animal trial., Setting: Hospital animal research laboratory., Subjects: Male Wistar rats challenged with intraperitoneal E. coli, O18ac K1, and treated 1 hr later with ceftriaxone and gentamicin., Interventions: Recombinant endotoxin neutralizing protein, 50 mg/kg, was administered to rats 1, 2, or 3 hrs after E. coli challenge; saline was administered to control animals., Measurements and Main Results: Quantitative bacteremia, 1 hr after challenge and before antibiotic administration, was not significantly different between treatment groups (range geometric mean 451 to 621 colony-forming units [cfu]/mL). The endotoxin concentration, measured immediately before recombinant endotoxin neutralizing protein administration, was significantly higher in animals sampled and treated at 2 hrs (geometric mean 260 EU/mL; 95% confidence interval 140 to 480 EU/mL), or 3 hrs (geometric mean 697 EU/mL; 95% confidence interval 307 to 1585 EU/mL) after E. coli challenge, compared with animals sampled and treated at 1 hr (geometric mean 17 EU/mL; 95% confidence interval 7 to 69 EU/ mL). Survival rate was significantly greater in rats treated with recombinant endotoxin neutralizing protein at 1 hr (23/27; p < .001) or 2 hrs (8/30; p < .01) after E. coli challenge than in controls (1/32)., Conclusion: Administration of recombinant endotoxin neutralizing protein delayed up to 2 hrs after challenge with E. coli improves survival in antibiotic-treated rats with Gram-negative sepsis.

Immunization of mothers during pregnancy may be an effective means of providing protection to infants during the first months of life against many pathogens. Previous studies have identified factors that influence the transfer of immunoglobulin across the placenta, including the time of vaccination during pregnancy and isotypes of specific immunoglobulins. By studying antibodies to Haemophilus influenzae type b polysaccharide (Hib-PS) in 26 pairs of maternal-cord sera obtained from unimmunized healthy women and 22 pairs of maternal-cord sera from women immunized with one of three different Hib vaccines, we have found that the immunoglobulin transfer is also dependent on the V region of antibodies. Anti-Hib-PS derived from the V kappa II gene "A2" was transferred about ten times more efficiently to the fetus than other anti-Hib-PS antibodies (20% vs 1-2%). It was found that antibodies derived from the A2 V kappa gene are primarily IgG whereas other antibodies are preferentially associated with the IgM isotype. The potential association between the antibody V region with preferential placental transfer should be considered for future studies involving maternal immunization.

Objective: To assess the benefit of a recombinant endotoxin neutralizing protein from Limulus polyphemus in treating Gram-negative bacterial sepsis in rabbits., Design: Prospective, blinded, controlled, laboratory trial., Setting: Animal research laboratory., Subjects: New Zealand White rabbits., Interventions: We established a rabbit model of Escherichia coli peritonitis and bacteremia, with high mortality rate, despite treatment with gentamicin and ceftriaxone. Twenty-five pairs of male New Zealand White rabbits were challenged intraperitoneally with E. coli O18ac K1 in 5% porcine mucin (mean 7 x 10(1) colony-forming units). All animals were treated with intravenous gentamicin (2.5 mg/kg) and ceftriaxone (100 mg/kg), and with either intravenous endotoxin neutralizing protein (50 mg/kg) or saline 1 hr after E. coli challenge., Measurements and Main Results: All animals were bacteremic 1 hr after challenge (mean 3.6 x 10(5) colony-forming units/mL). Animals in both groups developed tachycardia, hypotension, and acidosis (NS). Geometric mean serum endotoxin and tumor necrosis factor (TNF) concentrations were significantly ( p < .001) higher 1 hr after challenge compared with baseline prechallenge concentrations in both groups. From 1 to 2 hrs after challenge, endotoxin concentrations increased 2.5-fold in control animals (95% confidence interval = 13.1 to 32.9 endotoxin units/mL, p = .024), whereas endotoxin concentrations increased only 1.2-fold in endotoxin neutralizing protein-treated animals (95% confidence interval = 20.4 to 23.6 endotoxin units/mL, NS). TNF concentrations increased significantly (p < .001) in both groups from 1 to 2 hrs after challenge. Eighteen (72%) of 25 endotoxin neutralizing protein-treated animals vs. 11 (44%) of 25 controls survived 24 hrs (p = .032)., Conclusions: Treatment with endotoxin neutralizing protein had the following effects: a) the increase in serum endotoxin was blunted, but not TNF concentrations measured 1 hr after antibiotic treatment; and b) survival in rabbits with E. Coli sepsis was improved.

Respiratory syncytial virus (RSV) pneumonia is a well-recognized complication of bone marrow transplantation with a high mortality rate. We describe two patients who developed RSV pneumonia within the first 3 weeks following allogeneic bone marrow transplantation. These patients had significant oxygen requirements and radiographic infiltrates. Both were treated with aerosolized ribavirin and given a single 1.5 gm/kg dose of intravenous immune globulin containing high levels of RSV neutralizing activity (RSV-IG). Both patients showed subjective and objective improvement after RSV-IG, never required mechanical ventilation, and were discharged without an oxygen requirement within 2 weeks after therapy. RSV microneutralization activity was measured in serum and nasal secretions. Mean serum microneutralization activity increased from 2279 microneutralization units (Mu)/ml to 18082 Mu/ml after RSV-IG. Peak serum microneutralization activity achieved with RSV-IG was higher than that achieved in a series of other immunocompromised adults with RSV pneumonia given either multiple doses of standard IVIG or no immune globulin therapy. RSV-IG may be beneficial in the treatment of RSV pneumonia in severely immunocompromised patients.

Biodegradable polymeric devices intended to provide a viable route for single-dose vaccination were developed using controlled-release technology. One of the main challenges in the development of these devices was to overcome several water-mediated inactivation processes that occur in conventional polymeric systems. Our strategy was focused on the prevention of antigen exposure to environmental conditions. For this purpose a microencapsulation process was designed and optimized to provide an inert and insulated environment for the bioactive material inside controlled-release systems. Tetanus toxoid (TT) was used as a model antigen. The systems consist of core-wall microcapsule structures in which the antigenic protein is entrapped into oil-based cores of TT surrounded by outer polymer shells made of poly(D,L-lactide-co-glycolide), thus potentially protecting the bioactive material against deleterious conditions. Furthermore, using these microcapsules, pulses of immunochemically detected TT were programmed to release at two different times (3 and 7 weeks), as corroborated by in vitro release studies. The engineering of these specific antigen release properties was possible by careful selection of the copolymer composition and molecular weight. The final formulations were characterized with respect to morphology, structure, size distribution, and amount of immunochemically detected TT encapsulated. The new systems offer the potential to control the manner and timing of delivery. Over 92% of the TT released over a 63 day period from these microcapsules was immunochemically detected.

Objective: To evaluate maternal responses to Haemophilus influenzae type b (Hib) polysaccharide-tetanus protein conjugate (polyribosylribitol phosphate-tetanus or PRP-T) given to pregnant Gambian women, the transplacental transfer of antibody, and the effect of maternal immunization on infant responses to the vaccine., Design: An open, randomized immunogenicity study., Setting: A busy urban health center in The Gambia., Study Participants: A total of 451 pregnant women enrolled during the third trimester of pregnancy., Intervention: Study participants were randomized to three groups. In one group, mothers were given PRP-T during the third trimester and their infants were given PRP-T at 2, 3, and 4 months of age. In the second group, mothers received PRP-T and infants were given inactivated poliovirus vaccine. In the third group, mothers received meningococcal A and C vaccine, and their infants received PRP-T., Main Outcome Measures: Anti-PRP antibody measurements of maternal cord, and infant blood., Results: Vaccinated women had a marked increase in total anti-PRP antibody (geometric mean titer 9.0 micrograms/mL), which was greatest in women in their first or second pregnancy. Previous tetanus vaccination during the same pregnancy and high concentrations of antitetanus antibody were associated with lower anti-PRP responses. In infants of PRP-T recipients, cord blood anti-PRP IgG concentrations were 61% of simultaneous maternal concentrations. In vaccinated infants of vaccinated mothers, geometric mean anti-PRP antibody concentrations at birth, 2 months of age, and 5 months of age were 1.92, 0.35 and 2.84 micrograms/mL, respectively, while in vaccinated infants of unvaccinated mothers, the corresponding concentrations were 0.29, 0.12, and 3.91 micrograms/mL. At 2 months of age, 60% of infants of vaccinated mothers and 26% of infants of unvaccinated mothers had anti-PRP antibody concentrations considered to be protective (>0.15 micrograms/mL)., Conclusions: In areas where much invasive Hib disease occurs in infants younger than 6 months, maternal immunization may help to reduce the risk of Hib disease in infants too young for immunization.

Pertussis toxin (Ptx), the major toxin product of Bordetella pertussis, has potent immunologic effect including adjuvant effects on antibody responses and sensitization for anaphylaxis. In order to further define the effect of Ptx on the class and subclass of murine antibody response, we measured total and antigen specific IgG subclasses and IgE in Balb/c mice after primary and secondary immunization with tetanus toxoid (TT). Low doses of Ptx (100 ng) given intravenously at the time of primary immunization increased primary IgG1 and IgE anti-TT antibodies as well as total IgG1 and IgE concentrations compared to controls. The increase in IgG1 subclass and IgE response when Ptx was present during primary immunization was even more pronounced after secondary immunization with TT alone 3 weeks or 3 months later. Similar effects were noted after diphtheria toxoid immunization in the presence of Ptx. Administration of the anti IL-4 monoclonal antibody (11B11) suppressed the enhanced total and TT-specific IgE responses but not the enhanced IgG1 responses. The presence of low concentrations of Ptx during primary immunization primes for induction of IL-4 producing T-cell help which enhances IgGI and IgE responses to the primary exposure as well as to subsequent exposures of the antigen in the absence of Ptx. This phenomenon may have significance for the adjuvant activity of vaccines containing Ptx as well as for the immune response to natural pertussis.

An ELISA-based micro-neutralization (Nt) test in MRC-5 cells for titration of neutralizing antibodies against human cytomegalovirus (CMV) in human plasma and preparations of immune globulins was developed to eliminate microscopic reading of cytopathic effect (CPE), a process that is subjective and time consuming. Un-neutralized CMV from the Nt reaction and grown in MRC-5 cells as per the standard micro-Nt test was coated in the same plates by various methods and CMV antigen was quantified by polyclonal or monoclonal CMV antibodies. Optimal coating of plates with CMV antigen (100 TCID50 of virus grown on MRC-5 cells for 7 days) was obtained by freezing/thawing of virus infected MRC-5 cells in phosphate buffered saline, ph 7.2. The CMV antigen treated sequentially with CMV monoclonal antibody to late nuclear protein antigen, goat anti-mouse IgG3 alkaline phosphatase conjugate and phosphatase substrate gave an absorbance of 1 at 410 nm wavelength whereas uninfected MRC-5 cells treated under similar conditions did not show any absorbance. The optimal Nt reaction occurred at 37 degrees C for 1-2 h and was unaffected by complement. At 4 degrees C, CMV was inactivated in 1-2 h. The antibody titres were affected by the virus dose used in the Nt test over a range of 20 to 798 TCID50. When the titre was determined against a reference serum, the effect of virus dose on the Nt titre was reduced. Complete neutralization virus read microscopically correlated with ELISA absorbance of < 0.1. CPE produced by approximately 1 TCID50 of CMV showed an absorbance of 0.1 or more. The correlation coefficient (r) between Nt titres and CMV IgG antibodies determined by ELISA was 0.69 (P < 0.001) for 257 human plasma samples and 0.85 (P < 0.001) for 50 immune globulin preparations.

Non-phospholipid liposomes composed of dioxyethylene cetyl ether, cholesterol and oleic acid were evaluated as adjuvants with human vaccine antigens, tetanus toxoid (TT) and diphtheria toxoid (DT), in mice and rabbits. Antigens encapsulated in or mixed with liposomes elicited antitoxin levels similar to those elicited by antigens given with Freund's adjuvant or adsorbed onto aluminum phosphate. All liposomal antigen preparations, antigen given with Freund's adjuvant or adsorbed onto aluminum phosphate, elicited significantly higher IgG antibodies and antitoxin levels than soluble antigens in mice after a single injection and in rabbits after each of three injections. TT encapsulated in liposomes elicited sustained anti-TT IgG antibody levels in mice after a single injection as compared to TT mixed with liposomes. TT mixed with or encapsulated within liposomes containing monophosphoryl lipid A/squalene or squalene alone, as well as aluminum phosphate adsorbed TT elicited greater primary responses in mice than TT mixed with or encapsulated within plain liposomes. Liposomal TT preparations produced a slightly higher anamnestic response in mice than aluminum phosphate adsorbed TT. Subclass analysis of anti-TT antibodies showed that the majority of the antibodies belong to IgG1 subclass. Liposomal TT preparations, particularly those with encapsulated monophosphoryl lipid A/squalene or squalene alone, consistently elicited higher levels of anti-TT IgG2a and IgG2b than aluminum phosphate adsorbed or soluble TT. None of the preparations elicited IgG3 or IgM antibodies. It appears that non-phospholipid liposomes are as potent adjuvants as the currently employed adjuvant for human vaccines (aluminum phosphate) or a benchmark adjuvant for experimental immunology (Freund's adjuvant), and may be able to modulate the immune response towards the Th1 type.

Current United States (US) regulations for potency testing of vaccines containing adsorbed diphtheria and tetanus toxoids require in vivo toxin neutralization (TN) tests on the pooled sera of immunized guinea pigs. To reduce the number of animals required for testing, two in vitro tests have been evaluated, the Vero cell assay for diphtheria antitoxin and ELISA for IgG antibody to tetanus toxin; these have been correlated with in vivo TN tests. In the Vero cell method, diphtheria antitoxin titres of the guinea pig sera, obtained four weeks after immunization as per US potency requirements, were markedly dependent on the toxin dose level used in the assay. A toxin dose level termed the Lcd/1 dose (limit of cytopathic dose at a IU/ml) for Vero cells gave comparable estimates of antitoxin activity to the in vivo TN test performed at L+/1 dose of toxin. When lower dose levels of toxin were used in the Vero cells (Lcd/10 to Lcd/1000), diphtheria antitoxin levels in four weeks guinea pig sera were two to 11.7 times lower than with the Lcd/1 dose level. The most likely reason for these differences is that guinea pig sera a 4 weeks are of lower avidity than the equine antitoxin standard. Antibodies of low avidity bind antigen less well at low reactant concentrations. Therefore, to obtain similar estimates of diphtheria antitoxin in the Vero cell method and in vivo TN test, the use of toxin dose for the Vero cell method similar to that for the in vivo TN test is suggested. Another alternative, in which any dose of toxin may be used for the Vero cell method, is the use of a reference guinea pig serum (calibrated in IU/ml by the in vivo TN test at L+/1 level of toxin) that has similar avidity or similar immunization status as the test sera (i.e. 4 week serum). IgG antibodies to tetanus toxin in guinea pig sera were found early in the course of immunization when tetanus antitoxin could not be detected by TN test. Tetanus toxin IgG antibody levels of guinea pig sera calculated in IU/ml against an ELISA guinea pig reference serum (calibrated in IU/ml by TN test) depended upon the immunization status of the animals. To obtain similar estimates of tetanus antibodies in IU/ml by TN and ELISA, the ELISA reference guinea pig serum should have similar immunization status (and presumably similar avidity) as the test serum (i.e. six week serum). We propose that the Vero cell method and ELISA deserve further evaluation to determine whether they can replace in vivo TN tests for titration of diphtheria and tetanus antitoxins in the US potency test.

We have evaluated a guinea pig model for assessing the immunogenicity of Haemophilus influenzae type b (Hib) polysaccharide-protein conjugate vaccines, acellular pertussis vaccine and combination vaccines-consisting of tetanus toxoid (TT), diphtheria toxoid (DT), acellular pertussis vaccine and Hib-TT (Hib-T) conjugate vaccine. The model was based on the United States (US) potency test for TT and DT which requires injection of guinea pigs with a single dose of undiluted vaccine. Guinea pigs showed dose-dependent antibody responses to pertussis toxoid (PTxd) and filamentous haemagglutinin (FHA), two important components of acellular pertussis vaccine. Antibody response of guinea pigs to commercially available Hib conjugate vaccines qualitatively resembled those of human infants. Unconjugated polyribosylribitolphosphate (PRP) was not immunogenic; PRP-D conjugate produced a low antibody response, HbOC, PRP-T (Merieux) and Hib-T (MPHBL) produced a low response to the first dose and a strong anamnestic response to the booster dose. PRP-OMP uniquely produced a strong response after the first dose which was boosted by the second dose. In preliminary experiments, injection of guinea pigs with the combined vaccine formulations consisting of TT, DT, whole cell or acellular pertussis vaccine (Ptxd and FHA) and Hib-T conjugate showed that these vaccines were immunogenic when combined, with some effects on the antibody responses of certain components. This model for testing potency/immunogenicity of combined vaccines substantially reduces the number of animals needed to test each lot of vaccine. To reduce the use of animals in testing vaccines further, we propose the use of a Vero cell assay for titrating diphtheria antitoxin and ELISA for measuring IgG antibody to tetanus toxin. The guinea pig model may also be useful for evaluating combination vaccines.

An interlaboratory study was conducted to determine whether an enzyme-linked immunosorbent assay (ELISA) with an antigen preparation composed of various-sized fragments of Haemophilus influenzae type b polysaccharide conjugated to human serum albumin could be standardized across laboratories and whether the ELISA-derived results from different laboratories are equivalent to those obtained by the standard radioactive antigen binding assay (RABA) for quantitation of anti-H, influenzae type b polysaccharide antibodies. Twenty coded human serum samples were quantitated by ELISA in 11 laboratories and by RABA in 5 laboratories. The mean RABA-derived values served as the basis for all comparisons. While the overall correspondence of antibody values between the two methods was good, significant differences were found among some of the 11 ELISA data sets and among the mean RABA values. Seven laboratories generated higher ELISA antibody values for low-titered sera. Four laboratories generated antibody concentrations that were not statistically different between the two assay methods. The results therefore indicate that the ELISA can tolerate substantial variations in protocol, such as the use of different plates and different antibody reagents, without affecting the quantitation of serum antibodies. However, attention should be focused on low-titered sera, as some assay conditions may yield spurious results. This ELISA is a serologic assay which can serve as an alternative to the RABA for quantitation of antibodies to H. influenzae type h polysaccharide.

The stability of tetanus toxoid (TT) has been investigated for the purpose of enhancing its immunogenicity when encapsulated in TT-polylactide-co-glycolide (PLGA) microspheres. In this paper we describe our simulations of various potential inactivating events during microsphere processing and release of the antigen. These include: i) processing aqueous TT solutions in the presence of organic solvents, ii) exposing lyophilized TT to moisture, and iii) incubating vaccine with the degrading PLGA. At 37 degrees C, TT began to aggregate in solution a few hours after the addition of either methylene chloride or ethyl acetate to form a single oil-water interface. Similarly, exposure of the lyophilized vaccine to elevated humidity caused the antigen to lose solubility. Previous analysis of moisture-induced aggregates has revealed that formaldehyde, which is stored in labile linkages in the vaccine following its detoxification, is the precursor to the reactive species in the principal aggregation pathway [1]. Methods to inhibit this mechanism, such as blocking nucleophilic amino groups of TT with succinic anhydride, were verified. Succinylation of TT resulted in the incorporation of 10-fold greater antigenically active vaccine in PLGA microspheres relative to the unmodified TT following processing by double-emulsion/solvent evaporation with ethyl acetate, strongly suggesting the formaldehyde-mediated aggregation pathway also occurs during the deleterious conditions of microsphere processing. Incubation of solutions of TT in the presence of excess blank (unloaded) poly (D,L) lactide (mol. wt. 2000) microspheres led to a dramatic reduction in pH (approximately 3.2 units after one day at 45 degrees C) and simultaneous precipitation of TT. Stabilisation in the presence of the degrading polymer is likely to be the final obstacle before controlled-release preparations can be formulated to release antigenically active TT over extended time periods. Hence, mechanistic analyses as described here may be crucial for the development of effective single-dose vaccines.

Adjuvants help antigen to elicit an early, high and long-lasting immune response with less antigen, thus saving on vaccine production costs. In recent years, adjuvants received much attention because of the development of purified, subunit and synthetic vaccines which are poor immunogens and require adjuvants to evoke the immune response. With the use of adjuvants immune response can be selectively modulated to major histocompatibility complex (MHC) class I or MHC class II and Th1 or Th2 type, which is very important for protection against diseases caused by intracellular pathogens such as viruses, parasites and bacteria (Mycobacterium). A number of problems are encountered in the development and use of adjuvants for human vaccines. The biggest issue with the use of adjuvants for human vaccines, particularly routine childhood vaccines, is the toxicity and adverse side-effects of most of the adjuvant formulations. At present the choice of adjuvants for human vaccination reflects a compromise between a requirement for adjuvanticity and an acceptable low level of side-effects. Other problems with the development of adjuvants include restricted adjuvanticity of certain formulations to a few antigens, use of aluminum adjuvants as reference adjuvant preparations under suboptimal conditions, non-availability of reliable animal models, use of non-standard assays and biological differences between animal models and humans leading to the failure of promising formulations to show adjuvanticity in clinical trials. The most common adjuvants for human use today are still aluminum hydroxide and aluminum phosphate, although calcium phosphate and oil emulsions also have some use in human vaccinations. During the last 15 years much progress has been made on development, isolation and chemical synthesis of alternative adjuvants such as derivatives of muramyl dipeptide, monophosphoryl lipid A, liposomes, QS21, MF-59 and immunostimulating complexes (ISCOMS). Other areas in adjuvant research which have received much attention are the controlled release of vaccine antigens using biodegradable polymer microspheres and reciprocal enhanced immunogenicity of protein-polysaccharide conjugates. Biodegradable polymer microspheres are being evaluated for targeting antigens on mucosal surfaces and for controlled release of vaccines with an aim to reduce the number of doses required for primary immunization. Reciprocal enhanced immunogenicity of protein-polysaccharide conjugates will be useful for the development of combination vaccines.

Objective: To determine the efficacy of a murine anti-tumor necrosis factor (TNF) monoclonal antibody in the treatment of Escherichia coli peritonitis and sepsis in the rabbit., Design: Prospective, paired, randomized, blinded, controlled animal trial., Setting: Animal research laboratory., Subjects: Male New Zealand white rabbits., Interventions: Anesthetized rabbits were cannulated with indwelling femoral arterial and venous catheters. Peritonitis and sepsis were induced by intraperitoneal challenge using live E. coli O18ac bacteria. All animals were treated with gentamicin and ceftriaxone 1 hr after challenge. One group (prophylaxis experiment) consisting of ten rabbit pairs (the prophylaxis group), was treated with either murine anti-TNF monoclonal antibody or an equivalent volume of 5% albumin 3 hrs before E. coli challenge. A second group (therapeutic experiment) of 17 rabbit pairs, the treatment group, was also treated with murine anti-TNF monoclonal antibody or albumin control 1 hr after E. coli challenge., Measurements and Main Results: All animals were bacteremic 1 hr after challenge. Physiologic measures of sepsis (heart rate, mean arterial pressure, serum bicarbonate, and arterial pH) did not differ between control, prophylaxis, and treatment groups. Peak serum TNF concentration was significantly (p < .01) lower in animals receiving anti-TNF monoclonal antibody, in both the prophylaxis and treatment groups, than in control animals. The survival rate was not improved significantly in either the prophylaxis or treatment group., Conclusions: Prophylactic and therapeutic use of anti-TNF monoclonal antibody in a rabbit model of E. coli peritonitis and sepsis significantly lowers TNF concentrations but does not ameliorate the physiologic effects of sepsis and does not significantly improve survival.