A Novel Bacterial Protease Inhibitor Adjuvant in RBD-Based COVID-19 Vaccine Formulations Containing Alum Increases Neutralizing Antibodies, Specific Germinal Center B Cells and Confers Protection Against SARS-CoV-2 Infection in Mice.

In this work, we evaluated recombinant receptor binding domain (RBD)-based vaccine formulation prototypes with potential for further clinical development. We assessed different formulations containing RBD plus alum, AddaS03, AddaVax, or the combination of alum and U-Omp19: a novel Brucella spp. protease inhibitor vaccine adjuvant. Results show that the vaccine formulation composed of U-Omp19 and alum as adjuvants has a better performance: it significantly increased mucosal and systemic neutralizing antibodies in comparison to antigen plus alum, AddaVax, or AddaS03. Antibodies induced with the formulation containing U-Omp19 and alum not only increased their neutralization capacity against the ancestral virus but also cross-neutralized alpha, lambda, and gamma variants with similar potency. Furthermore, the addition of U-Omp19 to alum vaccine formulation increased the frequency of RBD-specific geminal center B cells and plasmablasts. Additionally, U-Omp19+alum formulation induced RBD-specific Th1 and CD8+ T-cell responses in spleens and lungs. Finally, this vaccine formulation conferred protection against an intranasal severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) challenge of K18-hACE2 mice.

Antigen phagocytosis by B cells is required for a potent humoral response.

Successful vaccines rely on activating a functional humoral response that results from promoting a proper germinal center (GC) reaction. Key in this process is the activation of follicular B cells that need to acquire antigens and to present them to cognate CD4 T cells. Here, we report that follicular B cells can phagocytose large antigen-coated particles, a process thought to be exclusive of specialized antigen-presenting cells such as macrophages and dendritic cells. We show that antigen phagocytosis by B cells is BCR-driven and mechanistically dependent on the GTPase RhoG. Using Rhog-/- mice, we show that phagocytosis of antigen by B cells is important for the development of a strong GC response and the generation of high-affinity class-switched antibodies. Importantly, we show that the potentiation effect of alum, a common vaccine adjuvant, requires direct phagocytosis of alum-antigen complexes by B cells. These data suggest a new avenue for vaccination approaches by aiming to deliver 1-3 µm size antigen particles to follicular B cells.

Different mechanisms for lead acetate, aluminum and cadmium sulfate in rat corpus cavernosum.

INTRODUCTION: Some heavy metals show adverse vascular and neurological effects, however, their effect on erection is underestimated. This study aims to investigate the effect of Pb, Cd and Al on erectile function and their potential mechanism of action in rats. METHODS: Measurement of intracavernosal pressure/mean arterial pressure (ICP/MAP) changes elicited by electrical stimulation of cavernous nerve in anesthetized rats treated with Pb-acetate, Al-sulfate, or Cd-sulfate acutely, and subacutely for 7 days. Serum creatinine, testosterone, TBARs, GSH levels and metal accumulation in corpus cavernosum were measured. RESULTS: Pb, Al and Cd significantly reduced ICP/MAP in rats after acute (2,10-2,10 and 1,3 mg/kg respectively) and sub-acute (3, 3, and 1mg/kg/day respectively) treatments. They selectively accumulated in the corpus cavernosum reaching 25.107 ± 2.081 µg/g wet weight for Pb, 1.029 ± 0.193 for Cd, 31.343 ± 1.991 for Al, compared to 7.084 ± 1.517, 0.296 ± 0.067, and 8.86 ± 1.115 as controls respectively. Serum creatinine levels were not altered. Cd and Al significantly reduced testosterone level to 0.483 ± 0.059 and 0.419 ± 0.037 ng/ml respectively compared to 0.927 ± 0.105 ng/ml as control. Aluminum elevated TBARs significantly by 27.843%. The acute anti-erectile action of Pb was blocked by non-selective NOS and GC inhibitors and potassium channel blocker. Lead also masked the potentiatory effect of l-arginine and diazoxide on ICP/MAP. No interaction with muscarinic or nicotinic modulators was observed. CONCLUSIONS: Pb, Cd and Al show anti-erectile effect independent on renal injury. They don not modulate cholinergic nor ganglionic transmission in corpus cavernosum. Pb may inhibit NO/cGMP/K+channel pathway. The effect of Cd and Al but not Pb seems to be hormonal dependent.

Effects of aluminium and bacterial lipopolysaccharide on oxidative stress and immune parameters in roach, Rutilus rutilus L.

Aluminium is used in diverse anthropogenic processes at the origin of pollution events in aquatic ecosystems. In the Champagne region (France), high concentrations of aluminium (Al) are detected due to vine-growing practices. In fish, little is known about the possible immune-related effects at relevant environmental concentrations. The present study analyzes the simultaneous effects of aluminium and bacterial lipopolysaccharide (LPS), alone and in combination, on toxicological biomarkers in the freshwater fish species Rutilus rutilus. For this purpose, roach treated or not with LPS were exposed to environmental concentrations of aluminium (100 µg/L) under laboratory-controlled conditions for 2, 7, 14 and 21 days. After each exposure time, we assessed hepatic lipoperoxidation, catalase activity, glutathione reductase activity and total glutathione content. We also analyzed cellular components related to the LPS-induced inflammatory response in possible target tissues, i.e. head kidney and spleen. Our results revealed a significant prooxidant effect in the liver cells and head kidney leukocytes of roach exposed to 100 µg of Al/L for 2 days. In liver, we observed more lipoperoxidation products and lower endogenous antioxidant activity levels such as glutathione reductase activity and total glutathione content. These prooxidant effects were associated with a higher oxidative burst in head kidney leukocytes, and they were all the more important in fish stimulated by LPS injection. These findings demonstrate that environmental concentrations of Al induce oxidative and immunotoxic effects in fish and are associated to an immunomodulatory process related to the inflammatory response.

Examination of the physical properties of Microcystis aeruginosa flocs produced on coagulation with metal salts.

Coagulation-flocculation (C-F) is a key barrier to cyanobacterial and algal cell infiltration in water treatment plants during seasonal blooms. However, the resultant cell floc properties, in terms of size, strength and density, which dominate under different coagulation conditions and govern cell removal, are not well understood. This paper investigated the floc properties produced during C-F of the cyanobacterium, Microcystis aeruginosa, under low and high doses of aluminium sulphate and ferric chloride coagulants and at different pH values, so as to promote charge neutralisation (CN) and sweep flocculation (SF) dominant conditions (or a combination of these). It was demonstrated that application of ferric chloride produced larger flocs that resulted in higher cell removal during jar testing. These flocs were also larger than those observed for natural organic matter (NOM) and kaolin, suggesting a role of algogenic organic matter (AOM) as an inherent bioflocculant. Under SF conditions, stronger flocs were produced; however, these had lower capacity for size recovery after exposure to high shear. Analysis of particle size distribution demonstrated that large scale fragmentation followed by erosion dominated for CN while erosion dominated under SF conditions. Overall, marked differences were observed dependent on the coagulation regime imposed that have implications for improving robustness of cell removal by downstream separation processes. While the cyanobacterium, M. aeruginosa, appeared to share general floc characteristics commonly observed for NOM and kaolin flocs, there were distinct differences in terms of size and strength, which may be attributed to AOM.

Bladder tissue permeability and transport modelling of intravesical alum, lidocaine hydrochloride, methylprednisolone hemisuccinate and mitomycin C.

The aims of this study were to assess the tissue permeability of the bladder and to characterize the transport of four drugs displaying different physico-chemical properties and commonly used in intravesical delivery, through porcine bladder. The transport of aluminium through porcine bladder was assessed by using a vertical static diffusion cell. Lidocaine hydrochloride, methylprednisolone hemisuccinate and mitomycin C were tested by using three different experimental setups, including vertical static diffusion cell, microdialyseur and lab-patented device. Penetration results on different experimental setups were homogenous suggesting dependency on physico-chemical characteristics of drug and subsequent interaction with bladder wall structure. Oppositely, permeation varied consistently with experimental setup characteristics (i.e., permeation surface, receptor fluid volume and hydrodynamic). Mathematical modelling of drug transport through bladder wall is proposed considering scarce literature on this route of administration. Practical outcome of this study could drive compounding optimization towards improvement of safety and efficacy in patient undergoing intravesical administration.

Astringency of foods may not be directly related to salivary lubricity.

UNLABELLED: Astringency, a tactile sensation felt in the mouth after exposure to various foods, is poorly understood. Our objective was to investigate the mechanism of astringency by determining if it was related to a loss of saliva's ability to lubricate mouth surfaces. We used 2 complementary approaches: (1) instrumentally determining if astringents decrease salivary lubricity and (2) determining if human subjects could perceive these changes. Instrumentally measured friction of saliva-tannin mixtures was greater than that of saliva-water mixtures, but these differences were not detectable by human subjects. Adding alum or acid to saliva did not increase friction measured either instrumentally or by human subjects rubbing the mixture between their thumb and fingers. Thus a loss of salivary lubricity is likely not a central mechanism of astringency. PRACTICAL APPLICATIONS: If astringency's mechanism can be elucidated, food producers will gain knowledge that will enable them to manufacture, alter, or treat the food in a way to retain its healthful attributes while minimizing the astringency. This knowledge will also benefit sensory scientists interested in the intensity perceptions of the sensations and will allow them to devise improved methods for the assessment of the attribute.

A unique role of the cholera toxin A1-DD adjuvant for long-term plasma and memory B cell development.

Adjuvants have traditionally been appreciated for their immunoenhancing effects, whereas their impact on immunological memory has largely been neglected. In this paper, we have compared three mechanistically distinct adjuvants: aluminum salts (Alum), Ribi (monophosphoryl lipid A), and the cholera toxin A1 fusion protein CTA1-DD. Their influence on long-term memory development was dramatically different. Whereas a single immunization i.p. with 4-hydroxy-3-nitrophenyl acetyl (NP)-chicken γ-globulin and adjuvant stimulated serum anti-NP IgG titers that were comparable at 5 wk, CTA1-DD-adjuvanted responses were maintained for >16 mo with a half-life of anti-NP IgG ∼36 wk, but <15 wk after Ribi or Alum. A CTA1-DD dose-dependent increase in germinal center (GC) size and numbers was found, with >60% of splenic B cell follicles hosting GC at an optimal CTA1-DD dose. Roughly 7% of these GC were NP specific. This GC-promoting effect correlated well with the persistence of long-term plasma cells in the bone marrow and memory B cells in the spleen. CTA1-DD also facilitated increased somatic hypermutation and affinity maturation of NP-specific IgG Abs in a dose-dependent fashion, hence arguing that large GC not only promotes higher Ab titers but also high-quality Ab production. Adoptive transfer of splenic CD80(+), but not CD80(-), B cells, at 1 y after immunization demonstrated functional long-term anti-NP IgG and IgM memory cells. To our knowledge, this is the first report to specifically compare and document that adjuvants can differ considerably in their support of long-term immune responses. Differential effects on the GC reaction appear to be the basis for these differences.

Naloxone and alum synergistically augment adjuvant activities of each other in a mouse vaccine model of Salmonella typhimurium infection.

Alum is the most commonly used adjuvant for human vaccination but is a poor inducer of cell mediated immunity and T helper 1 (Th1) responses. We have previously shown that naloxone (NLX), which is a general opioid antagonist, acts as an effective adjuvant in enhancing vaccine-induced cellular immunity and Th1 immune responses. Here, we tested the efficacy of an alum-NLX mixture, as a new adjuvant, in the induction of humoral and cellular immunity in response to endotoxin-removed lysate (ERL) of Salmonella typhimurium (S. typhimurium) as a model vaccine. BALB/c mice were divided into five vaccination groups. Mice in the experimental groups received either the ERL vaccine alone or in combination with the adjuvant alum, NLX or the alum-NLX mixture. Mice in the negative control group received phosphate-buffered saline. All mice were immunized on days 0 and 7. Two weeks after the last immunization, immune responses to S. typhimurium were assessed. Our results indicate that including the alum-NLX mixture as an adjuvant during vaccination increased the ability of the ERL vaccine to enhance lymphocyte proliferation, shifted the immune response toward a Th1 profile and increased S. typhimurium-specific IgG, IgG2a and the ratio of IgG2a to IgG1. This resulted in improved protective immunity against S. typhimurium. In conclusion, administering an alum-NLX mixture adjuvant in combination with the ERL vaccine enhances both humoral and cellular immunity, and shifts the immune response to a Th1 pattern.

Evidence supporting a biological role for aluminum in chromatin compaction and epigenetics.

Averaging 8.1% (w/w) of the earth's crust, aluminum is the most highly abundant metal in our biosphere, yet has long been thought to serve no essential biological function. In aqueous solutions, aluminum salts and hydroxides are exceptionally potent aggregators of biological molecules, often coalescing molecular species to the point that they precipitate out of solution. A biological function for aluminum is proposed in which this abundant, high charge density metal cation has a significant role in biomolecular compaction. Sometimes, molecules ectopically aggregated by aluminum are associated with pathological conditions. The data further suggests that a specific consequence of 'aluminum biocompaction' may be particularly important in the condensation of A+T-rich chromatin domains, and in silencing the expression of specific kinds of genetic information.

Alum application to improve water quality in a municipal wastewater treatment wetland: effects on macrophyte growth and nutrient uptake.

Application of low doses of alum to treatment wetlands to reduce elevated outflow winter phosphorus concentrations were tested in mesocosms vegetated with either Typhadomingensis, Schoenoplectus californicus, or submerged aquatic vegetation (SAV) (Najas guadalupensis-dominated). Alum was pumped to experimental units at a rate of 0.91 g Al m(-2) d(-1) and water quality monitored for 3 months. The alum application significantly improved the outflow water quality and overall the growth of the plants was unaffected by the alum application. Biomass and growth varied between species and through time, but no significant effects of alum application were detected. The concentrations of nutrients and mineral elements in the aboveground tissues differed between species and over time, but only the concentration of Al in plant tissue was increased by alum additions. The concentration of Al was 50-fold higher in alum-treated SAV as compared to the control, and in Typha and Schoenoplectus the concentrations were 4- and 2-fold, higher, respectively. The N/P ratios in the plant tissues were low (<10) suggesting that their growth and biomass was limited by nitrogen. The research suggests that a continuous or seasonal low-dosage alum application to treatment wetlands provides an effective tool to maintain discharge concentrations within permitted values during the inefficient winter treatment times. We suggest that the use of alum should be restricted to treatment wetland areas dominated by emergent vegetation as the effects of the elevated Al concentrations in SAV needs further study.

Modulation of the SecY channel permeability by pore mutations and trivalent cations.

The SecY channel serves to transport proteins across the bacterial inner membrane. The closed channel is impermeable to small molecules by means of a plug domain and a hydrophobic pore, consisting of six conserved isoleucine residues. The substitution of these isoleucines by asparagine leads to the selective conductance of small monovalent anions, especially chloride. In this addendum, we show that replacement of the isoleucine residues by bulky phenylalanine also leads to an increased chloride conductance, suggesting that hydrophobicity of the pore is not the sole determinant for maintaining channel impermeability. Instead, incubation of the membrane with the trivalent cation Al(3+) dramatically increases Cl(-) transport across the wild type SecY channel, suggesting that surface charge density around the SecY pore plays a significant role during the process of chloride conductance.

Mechanism of action of clinically approved adjuvants.

Aluminum-containing adjuvants continue to be the most widely used adjuvants for human use. In the last year a major breakthrough has been the realization that alum adjuvant triggers an ancient pathway of innate recognition of crystals in monocytes and triggers them to become immunogenic dendritic cells, nature's adjuvant. This recognition can occur directly, via the triggering of the NALP3 inflammasome by alum crystals, or indirectly through release of the endogenous danger signal uric acid. It is also clear now that adjuvants trigger the stromal cells at the site of injection, leading to the necessary chemokines that attract the innate immune cells to the site of injection. How exactly these pathways interact remains to be determined.

The impact of alum coagulation on the character, biodegradability and disinfection by-product formation potential of reservoir natural organic matter (NOM) fractions.

Natural Organic Matter (NOM) from Myponga Reservoir, South Australia, was separated into four organic fractions based on their hydrophobic and hydrophilic properties using a sequence of non-ionic and ionic resins. NOM fractions were isolated for the purpose of determining the impact of alum coagulation on removal of these fractions in conventional water treatment, and their potential as precursors in the formation of disinfection by-products (DBP) and in supporting microbial growth. The NOM comprised VHA (very hydrophobic acids), SHA (slightly hydrophobic acids), CHA (charged hydrophilics) and NEU (neutral hydrophilics) fractions. These fractions were then jar tested with alum using low (50 mg/L), operational (100 mg/L) and very high (200 mg/L) doses to assess the removal capacities for these fractions in a conventional treatment plant. High-performance size exclusion chromatography-UV-DOC (HPSEC-UV-DOC) revealed that alum removed more of the hydrophobic and higher molecular weight components of NOM, but less of the NEU fraction and lower molecular weight components of NOM. Determination of biodegradable dissolved organic carbon (BDOC) indicated that the NEU fraction had the highest biodegradability, followed by the CHA, SHA and VHA fractions. The VHA fraction had the highest total-trihalomethane formation potential (t-THMFP), followed by NEU, SHA and CHA. The NOM not removed by alum coagulation had the potential to support microbial growth (NEU fraction), and disinfection by-product (DBP) formation (VHA and NEU fractions). To obtain treated water with lower overall residual NOM, other treatment methods would need to be applied in addition to alum coagulation in order to reduce the concentration of the neutral fraction.

Behavior of aluminum in aluminum welders and manufacturers of aluminum sulfate--impact on biological monitoring.

OBJECTIVES: The suitability of determining aluminum in serum or urine as a form of biological monitoring was critically assessed. METHODS: Airborne and internal aluminum exposure was assessed for 12 aluminum welders in a shipyard and 5 manufacturers of aluminum sulfate. Particles were characterized with X-ray diffraction and scanning electron microscopy. Aluminum in air and biological samples was analyzed using electrothermal atomic absorption spectrometry. Basic toxicokinetic features were inferred from the data. RESULTS: The mean 8-hour time-weighted average concentration of aluminum was 1.1 (range 0.008-6.1) mg/m(3) for the shipyard and 0.13 (range 0.02-0.5) mg/m(3) for the aluminum sulfate plant. Welding fume contained aluminum oxide particles <0.1 microm in diameter and their agglomerates, whereas bauxite and aluminum sulfate particles ranged from 1 to 10 microm in diameter. The shipyard welders' mean postshift serum and urinary concentrations of aluminum (S-Al and U-Al, respectively) were 0.22 and 3.4 micromol/l, respectively, and the aluminum sulfate workers' corresponding values were 0.13 and 0.58 micromol/l. Between two shifts, the welders' S-Al concentration decreased by about 50% (P<0.01), but their U-Al concentration did not change (P=0.64). No corresponding temporal changes occurred among the aluminum sulfate workers. After aluminum welding at the shipyard had ceased, the median S-Al concentration decreased by about 50% (P=0.007) within a year, but there was no change (P=0.75) in the corresponding U-Al concentration. CONCLUSIONS: About 1% of aluminum in welding fume appears to be rapidly absorbed from the lungs, whereas an undetermined fraction is retained and forms a lung burden. A higher fractional absorption of aluminum seems possible for aluminum sulfate workers without evidence of a lung burden. After rapid absorption, aluminum is slowly mobilized from the lung burden and dominates the S-Al and U-Al concentrations of aluminum welders. For kinetic reasons, S-Al or U-Al concentrations cannot be used to estimate the accumulation of aluminum in the target organs of toxicity. However, using U-Al analysis to monitor aluminum welders' lung burden seems practical.

Review: novel nonviral delivery approaches for interleukin-12 protein and gene systems: curbing toxicity and enhancing adjuvant activity.

It has become increasingly apparent that the ability to generate an optimal host immune response requires effective cross talk between the innate and adaptive components of the immune system. Pro-inflammatory cytokines, in particular those that can induce a danger signal, often called signal 3, are crucial in this role of initiating and augmenting the presentation of exogenous antigen to T cells by dendritic cells. Interleukin-12 (IL-12) in particular has been defined as a "signal 3" cytokine required for the antigen cross priming. Given this unique interactive function, a significant amount of work has been performed to define possible therapeutic applications for IL-12. Systemic IL-12 administration can clearly act as a potent adjuvant for postvaccination T cell responses in a variety of diseases. As an example, in the cancer setting, systemic IL-12 is capable of suppressing tumor growth, metastasis, and angiogenesis in vivo. IL-12, however, has been associated with significant dose- and schedule-dependent toxicity in early clinical trials, results that have proven to be a major obstacle to its clinical application. Recent research has focused on decreasing the toxicity of IL-12 using different delivery approaches, including virus-based and gene-modified cell-based delivery. Although effective, these approaches also have limitations, including the generation of neutralizing antibodies, in addition to lacking the simplicity and versatility required for universal clinical application. Thus, there is a significant interest in the development of alternative delivery approaches for IL-12 administration that can overcome these issues. Several nonviral delivery approaches for IL-12 protein or gene expression vectors are being defined, including alum, liposomes, and polymer-based delivery. These developing approaches have shown promising adjuvant effects with significantly lessened systemic toxicity. This article discusses the potential capabilities of these nonvirus-based IL-12 delivery systems in different disease settings, including allergy, infection, and cancer.

GM1 binding-deficient exotoxin is a potent noninflammatory broad spectrum intradermal immunoadjuvant.

Intradermal (i.d.) immunization is a promising route of vaccine administration. Suitable i.d. adjuvants are important to increase vaccine efficacy in poorly responding populations such as the elderly or for dose-sparing strategies in the face of vaccine shortages. Bacterial exotoxins, such as Escherichia coli heat-labile enterotoxin (LT), exert strong immunostimulatory effects through binding to monosialoganglioside (GM1) cell surface receptors; however, injection is hampered by local inflammation. We demonstrate that the injection of LT formulations deficient in GM1 binding by mutation (LT(G33D)) or in vitro ligand coupling does not cause localized edema and inflammation in mice, yet these formulations retain potent adjuvant activity by enhancing functional Ab and cellular immune responses to coadministered Ags. Complete protection against in vivo lethal tetanus toxin challenge and the induction of Ag-specific CTL responses capable of killing target cells in vivo indicated in vivo efficacy of the induced immune responses. LT(G33D) proved superior to standard alum adjuvant regarding the magnitude and breadth of the induced immune responses. Immunizations in complex ganglioside knockout mice revealed a GM1-independent pathway of LT adjuvanticity. Immunostimulation by i.d. LT(G33D) is explained by its ability to induce migration of activated APCs to the proximal draining lymph nodes. LT(G33D) is a promising candidate adjuvant for human trials of parenteral vaccines in general and for current i.d. vaccine development in particular.

Removal of PCR inhibitors from soil DNA by chemical flocculation.

Extracting high-purity DNA directly from soil has become essential for the study of microorganisms in environmental samples. However, many soils contain compounds that inhibit enzymes involved in manipulating DNA. In this study, chemical flocculation using multivalent cations was investigated as a potential method for eliminating soil-based inhibitors during the extraction process. The addition of AlNH(4)(SO(4))(2) during extraction significantly reduced the co-purification of PCR inhibitors with minimal loss of DNA yield.

The partially degraded lipopolysaccharide of Burkholderia cepacia and ornithine-containing lipids derived from some Gram-negative bacteria are useful complex lipid adjuvants.

The partially degraded lipopolysaccharide of Burkholderia cepacia (LPSdegr) and the ornithine-containing lipids were purified from some bacteria. The substances were developed as complex lipid adjuvants, because they have weak toxicity and are able to activate the immune systems of the living body. After various toxoid antigens such as pertussis toxoid, diphtheria toxoid and tetanus toxoid were mixed with the complex lipid adjuvants, the mixtures were administered to mice subcutaneously. Antitoxoid IgG antibody titers in the serum were measured several times over 3 months. The efficacy of the LPSdegr as adjuvant was almost as high as that of the ornithine-containing lipids, and it was almost equal to that of the aluminum hydroxide adjuvant (Alum), which is generally used as a vaccine adjuvant.

Effects of alum adjuvant or a booster dose on immunogenicity during clinical trials of group B streptococcal type III conjugate vaccines.

Phase 1 and 2 clinical trials of group B streptococcal (GBS) capsular polysaccharide (CPS)-protein conjugate vaccines in healthy adults have demonstrated their safety and improved immunogenicity compared with uncoupled CPSs. Two recent trials sought to determine (i) whether adsorption of conjugate vaccine to aluminum hydroxide would improve immunogenicity and (ii) whether the CPS-specific immunoglobulin G (IgG) response could be boosted by administration of a second dose. Adsorption of GBS type III CPS-tetanus toxoid (III-TT) conjugate vaccine to alum did not improve the immune response to a 12.5-microg dose in healthy adult recipients. Four weeks after vaccination, the geometric mean antibody concentrations (GMCs) for the 15 recipients of III-TT with or without alum were 3.3 and 3.6 microg/ml, respectively. In the second trial, 36 healthy adults vaccinated previously with GBS III-TT conjugate were given a second 12.5-microg dose 21 months later. At 4 weeks after the second dose, the GMCs of type III CPS-specific IgG were similar to those measured 4 weeks after the primary vaccination, suggesting a lack of a booster response. However, 8 (22%) of the 36 participants who had undetectable III CPS-specific IgG (<0.05 microg/ml) before the first dose of III-TT conjugate exhibited a booster response to the second dose, with a fourfold-greater GMC of type III CPS-specific IgG than after the initial immunization. These results suggest that prior natural exposure to type III GBS or a related antigen may be responsible for the brisk IgG response to CPS noted in most adults after vaccination. However, a second dose of GBS III-TT conjugate vaccine may be required for adults whose initial CPS-specific IgG concentrations are very low and would also restore the initial peak-specific III CPS-IgG in responders to previous vaccination.