This open-label, phase I/II study investigated the safety and efficacy of epratuzumab, a humanised anti-CD22 monoclonal antibody, in the treatment of patients with active primary Sjögren's syndrome (pSS). Sixteen Caucasian patients (14 females/2 males, 33-72 years) were to receive 4 infusions of 360 mg/m2 epratuzumab once every 2 weeks, with 6 months of follow-up. A composite endpoint involving the Schirmer-I test, unstimulated whole salivary flow, fatigue, erythrocyte sedimentation rate (ESR), and immunoglobulin G (IgG) was devised to provide a clinically meaningful assessment of response, defined as a>> or = 20% improvement in at least two of the aforementioned parameters, with>> or = 20% reduction in ESR and/or IgG considered as a single combined criterion. Fourteen patients received all infusions without significant reactions, 1 patient received 3, and another was discontinued due to a mild acute reaction after receiving a partial infusion. Three patients showed moderately elevated levels of Human anti-human (epratuzumab) antibody not associated with clinical manifestations. B-cell levels had mean reductions of 54% and 39% at 6 and 18 weeks, respectively, but T-cell levels, immunoglobulins, and routine safety laboratory tests did not change significantly. Fifty-three percent achieved a clinical response (at>> or = 20% improvement level) at 6 weeks, with 53%, 47%, and 67% responding at 10, 18, and 32 weeks, respectively. Approximately 40%-50% responded at the>> or = 30% level, while 10%-45% responded at the>> or = 50% level for 10-32 weeks. Additionally, statistically significant improvements were observed in fatigue, and patient and physician global assessments. Further, we determined that pSS patients have a CD22 over-expression in their peripheral B cells, which was downregulated by epratuzumab for at least 12 weeks after the therapy. Thus, epratuzumab appears to be a promising therapy in active pSS, suggesting that further studies be conducted.

Cholera, caused by Vibrio cholerae, is a noninvasive dehydrating enteric disease with a high mortality rate if untreated. Infection with V. cholerae elicits long-term protection against subsequent disease in countries where the disease is endemic. Although the mechanism of this protective immunity is unknown, it has been hypothesized that a protective mucosal response to V. cholerae infection may be mediated by anamnestic responses of memory B cells in the gut-associated lymphoid tissue. To characterize memory B-cell responses to cholera, we enrolled a cohort of 39 hospitalized patients with culture-confirmed cholera and evaluated their immunologic responses at frequent intervals over the subsequent 1 year. Memory B cells to cholera antigens, including lipopolysaccharide (LPS), and the protein antigens cholera toxin B subunit (CTB) and toxin-coregulated pilus major subunit A (TcpA) were enumerated using a method of polyclonal stimulation of peripheral blood mononuclear cells followed by a standard enzyme-linked immunospot procedure. All patients demonstrated CTB, TcpA, and LPS-specific immunoglobulin G (IgG)and IgA memory responses by day 90. In addition, these memory B-cell responses persisted up to 1 year, substantially longer than other traditional immunologic markers of infection with V. cholerae. While the magnitude of the LPS-specific IgG memory B-cell response waned at 1 year, CTB- and TcpA-specific IgG memory B cells remained significantly elevated at 1 year after infection, suggesting that T-cell help may result in a more durable memory B-cell response to V. cholerae protein antigens. Such memory B cells could mediate anamnestic responses on reexposure to V. cholerae.

Three types of receptor for the Fc (constant) region of human immunoglobulin G have been described; FcRI, a high-affinity (Ka approximately equal to 10(8) M-1) receptor expressed on monocytes; FcRII (CD32), a low-affinity (Ka approximately equal to 10(6) M-1) receptor expressed on B cells, granulocytes, macrophages and platelets; and FcRIII (CD16, FcRIo), a low-affinity receptor expressed on macrophages, neutrophils, eosinophils, natural killer cells and a subset of T cells believed to comprise the suppressor cells. Anti-CD16 antibodies block natural killer-cell mediated antibody dependent cellular cytotoxicity (ADCC). Binding of aggregated IgG to CD16 on natural killer cells leads to the expression of lymphocyte activation antigens, mediator release, morphological changes and lytic activity. We report here the isolation of a complementary DNA clone encoding CD16 determinants which gave rise to IgG binding of the expected affinity and subtype specificity in COS cells, and which proved to encode a phospholipid anchored protein. A single messenger RNA transcript was found in all positive RNA samples, and N-glycanase treatment showed the form found in COS cells was identical to the form present on peripheral blood mononuclear cells (PBMCs). We also show that CD16 is most closely related to the alpha-form of the murine IgG 2b/1 receptor and propose that extracellular contacts mediate the signal initiated by IgG binding.

Voltage-gated Na(+) channels (VGSCs) exist as macromolecular complexes containing a pore-forming alpha subunit and one or more beta subunits. The VGSC alpha subunit gene family consists of 10 members, which have distinct tissue-specific and developmental expression profiles. So far, four beta subunits (beta1-beta4) and one splice variant of beta1 (beta1A, also called beta1B) have been identified. VGSC beta subunits are multifunctional, serving as modulators of channel activity, regulators of channel cell surface expression, and as members of the immunoglobulin superfamily, cell adhesion molecules (CAMs). beta subunits are substrates of beta-amyloid precursor protein-cleaving enzyme (BACE1) and gamma-secretase, yielding intracellular domains (ICDs) that may further modulate cellular activity via transcription. Recent evidence shows that beta1 regulates migration and pathfinding in the developing postnatal CNS in vivo. The alpha and beta subunits, together with other components of the VGSC signaling complex, may have dynamic interactive roles depending on cell/tissue type, developmental stage, and pathophysiology. In addition to excitable cells like nerve and muscle, VGSC alpha and beta subunits are functionally expressed in cells that are traditionally considered nonexcitable, including glia, vascular endothelial cells, and cancer cells. In particular, the alpha subunits are up-regulated in line with metastatic potential and are proposed to enhance cellular migration and invasion. In contrast to the alpha subunits, beta1 is more highly expressed in weakly metastatic cancer cells, and evidence suggests that its expression enhances cellular adhesion. Thus, novel roles are emerging for VGSC alpha and beta subunits in regulating migration during normal postnatal development of the CNS as well as during cancer metastasis.

OBJECTIVE

To investigate the safety, tolerability, and pharmacokinetics of the anti-tumor necrosis factor-alpha monoclonal antibody infliximab in subjects with intravenous immunoglobulin (IVIG)-resistant Kawasaki disease (KD).

METHODS

We conducted a multicenter, randomized, prospective trial of second IVIG infusion (2 g/kg) versus infliximab (5 mg/kg) in 24 children with acute KD and fever after initial treatment with IVIG. Primary outcome measures were the safety, tolerability, and pharmacokinetics of infliximab. Secondary outcome measures were duration of fever and changes in markers of inflammation.

RESULTS

Study drug infusions were associated with cessation of fever within 24 hours in 11 of 12 subjects treated with infliximab and in 8 of 12 subjects retreated with IVIG. No infusion reactions or serious adverse events were attributed to either study drug. No significant differences were observed between treatment groups in the change from baseline for laboratory variables, fever, or echocardiographic assessment of coronary arteries.

CONCLUSIONS

Both infliximab and a second IVIG infusion were safe and well tolerated in the subjects with KD who were resistant to standard IVIG treatment. The optimal management of patients resistant to IVIG remains to be determined.

We studied the potential inhibitory effect of Lactobacillus casei strain Shirota (from the fermented milk product Yakult [Yakult Ltd., Tokyo, Japan]) on Helicobacter pylori by using (i) in vitro inhibition assays with H. pylori SS1 (Sydney strain 1) and nine H. pylori clinical isolates and (ii) the in vivo H. pylori SS1 mouse model of infection over a period of 9 months. In vitro activity against H. pylori SS1 and all of the clinical isolates was observed in the presence of viable L. casei strain Shirota cells but not in the cell-free culture supernatant, although there was profound inhibition of urease activity. In vivo experiments were performed by oral administration of L. casei strain Shirota in the water supply over a period of 9 months to 6-week-old C57BL/6 mice previously infected with H. pylori SS1 (study group; n = 25). Appropriate control groups of H. pylori-infected but untreated animals (n = 25) and uninfected animals given L. casei strain Shirota (n = 25) also were included in the study. H. pylori colonization and development of gastritis were assessed at 1, 2, 3, 6, and 9 months postinfection. A significant reduction in the levels of H. pylori colonization was observed in the antrum and body mucosa in vivo in the lactobacillus-treated study group, as assessed by viable cultures, compared to the levels in the H. pylori-infected control group. This reduction was accompanied by a significant decline in the associated chronic and active gastric mucosal inflammation observed at each time point throughout the observation period. A trend toward a decrease in the anti-H. pylori immunoglobulin G response was measured in the serum of the animals treated with lactobacillus, although this decrease was not significant.

To characterize the distribution of Zaire ebolavirus (ZEBOV) infection within the 3 bat species (Epomops franqueti, Hypsignathus monstrosus, and Myonycteris torquata) that are possible reservoirs, we collected 1390 bats during 2003-2006 in Gabon and the Republic of the Congo. Detection of ZEBOV immunoglobulin G (IgG) in 40 specimens supports the role of these bat species as the ZEBOV reservoirs. ZEBOV IgG prevalence rates (5%) were homogeneous across epidemic and nonepidemic regions during outbreaks, indicating that infected bats may well be present in nonepidemic regions of central Africa. ZEBOV IgG prevalence decreased, significantly, to 1% after the outbreaks, suggesting that the percentage of IgG-positive bats is associated with virus transmission to other animal species and outbreak appearance. The large number of ZEBOV IgG-positive adult bats and pregnant H. monstrosus females suggests virus transmission within bat populations through fighting and sexual contact. Our study, thus, helps to describe Ebola virus circulation in bats and offers some insight into the appearance of outbreaks.

The MHC Class I-related receptor, FcRn, transports antibodies of the immunoglobulin G (IgG) class within and across a diverse array of different cell types. Through this transport, FcRn serves multiple roles throughout adult life that extend well beyond its earlier defined function of transcytosing IgGs from mother to offspring. These roles include the maintenance of IgG levels and the delivery of antigen in the form of immune complexes to degradative compartments within cells. Recent studies have led to significant advances in knowledge of the intracellular trafficking of FcRn and (engineered) IgGs at both the molecular and cellular levels. The engineering of FcRn-IgG (or Fc) interactions to generate antibodies of increased longevity represents an area of active interest, particularly in the light of the expanding use of antibodies in therapy. The strict pH dependence of FcRn-IgG interactions, with binding at pH 6 that becomes essentially undetectable as near neutral pH is approached, is essential for efficient transport. The requirement for retention of low affinity at near neutral pH increases the complexity of engineering antibodies for increased half-life. Conversely, engineered IgGs that have gained significant binding for FcRn at this pH can be potent inhibitors of FcRn that lower endogenous IgG levels and have multiple potential uses as therapeutics. In addition, molecular studies of FcRn-IgG interactions indicate that mice have limitations as preclinical models for FcRn function, primarily due to cross-species differences in FcRn-binding specificity.

Practically any oligopeptide can be exposed on the surface of the bacteriophage capsid by fusion to the major coat protein of filamentous bacteriophages. A phage expressing a particular peptide tag can be selected from a mixture of tens of millions of clones, exposing oligopeptides of random sequence, by affinity purification with a protein ligand. In this respect, pVIII can be used as an alternative and complement to the exposition vectors based on the product of gene III (pIII). We have constructed a phagemid vector that contains gene VIII under the control of the pLac promoter. This vector can be conveniently used to construct libraries of oligopeptides with a random amino acid sequence. An antipeptide monoclonal antibody was used to affinity-purify phagemids exposing oligopeptides which can interact with the monoclonal antibody. DNA sequencing of the amino terminus of gene VIII of the recovered clones predicts the synthesis of hybrid proteins whose aminoterminal amino acid sequence is related to that of the oligopeptide used to raise the antibody. In other words, only oligopeptides that bind a very small portion of the immunoglobulin G surface are affinity-purified by this method, implying that the antigen binding site possesses molecular properties that renders it much stickier than the remainder of the molecule.

Numerous in vitro and in vivo studies in both animals and patients with asthma have shown that interleukin (IL)-9 is an important inflammatory mediator in asthma. To examine the effects of IL-9 antagonism on airway inflammation, ovalbumin-sensitized BALB/c mice were intravenously given anti-IL-9 antibody or an isotype-matched control antibody 30 minutes before challenge with aerosolized ovalbumin. Airway response to methacholine was measured, and samples of bronchoalveolar lavage fluid (BALF) were obtained 24 hours after the last antigen challenge. Lung tissue was harvested and examined histopathologically. After ovalbumin challenge, there were significant increases in airway hyperreactivity, the numbers of inflammatory cells in lung, and IL-4, IL-5, and IL-13 production in BALF. Treatment with anti-IL-9 antibody significantly prevented airway hyperreactivity in response to methacholine inhalation. Blockade of IL-9 reduced the numbers of eosinophils (0.3 +/- 0.1 x 10(5) and 23.6 +/- 0.5 x 10(5)/ml, anti-IL-9 antibody/control immunoglobulin G) and lymphocytes (0.2 +/- 0.2 x 10(5) and 0.8 +/- 0.1 x 10(5)/ml) in BALF. Anti-IL-9 antibody treatment also reduced the concentrations of IL-4 (from 70.6 +/- 4.6 to 30.8 +/- 5.2 pg/ml), IL-5 (from 106.4 +/- 12 to 54.4 +/- 6.6 pg/ml), and IL-13 (from 44.2 +/- 7.6 to 30.1 +/- 5.5 pg/ml) in BALF. Macrophage-derived cytokine expression in the airways was also decreased by IL-9 blockade. Taken together, our findings emphasize the importance of IL-9 in the pathogenesis of asthma and suggest that blockade of IL-9 may be a new therapeutic strategy for bronchial asthma.

Non-immune kittens passively immunized with feline serum containing high-titered antibodies reactive with feline infectious peritonitis virus (FIPV) developed a more rapid disease after FIPV challenge than did kittens pretreated with FIPV antibody-negative serum. Antibody-sensitized, FIPV challenged--kittens developed earlier clinical signs (including pyrexia, icterus, and thrombocytopenia) and died more rapidly than did non-sensitized, FIPV-challenged kittens. Mean survival time in sensitized kittens was significantly (P less than 0.05) reduced compared to non-sensitized kittens (mean +/- SEM, 10.0 +/- 0.6 days vs. 28.8 +/- 8.3 days, respectively). Lesions induced included fibrinous peritonitis, disseminated pyogranulomatous inflammation and necrotizing phlebitis and periphlebitis. FIPV antigen, immunoglobulin G, complement (C3) and fibrinogen were demonstrated in lesions by immunofluorescence microscopy. The pathogenesis of dengue hemorrhagic fever (DHF) in persons bears striking resemblance to that of FIP in experimental kittens. In both FIP and DHF, non-neutralizing antibody may promote acute disease by enhancement of virus infection in mononuclear phagocytes or by formation of immune complexes, activation of complement and secondary vascular disturbances.

Pneumolysin, a membrane-damaging toxin, is known to activate the classical complement pathway. We have shown that 1 microgram ml-1 of pneumolysin can activate complement, which is a much lower level than observed previously. We have identified two distinct regions of pneumolysin which show homology with a contiguous sequence within acute-phase proteins, including human C-reactive protein (CRP). Site-directed mutagenesis of the pneumolysin gene was used to change residues common to pneumolysin and CRP. Some of the modified toxins had a reduced ability both to activate complement and bind antibody. We suggest that the ability of pneumolysin to activate complement is related to its ability to bind the Fc portion of immunoglobulin G.

The in vivo properties of an immunoglobulin isotype-switch family of monoclonal antibodies specific for the polysaccharide capsule of Cryptococcus neoformans were examined in a murine model of cryptococcosis. Subclass-switch variants were isolated by sequential sublining of an immunoglobulin G subclass 1 (IgGGGGGGGimmunoglobulin isotype. Despite apparent efficacy with regard to reduction in the numbers of yeast cells in the lung and spleen, the results showed no improvement in survival from murine cryptococcosis. Our results indicate that passive immunization produces a modest effect on the course of murine cryptococcosis in tissues other than brain. However, under the experimental conditions used, such treatment does not have a measurable impact on the ultimate outcome of the infection.

Alveolar macrophages are the initial phagocytic cells that encounter foreign material and particulates deposited in the terminal airways. We have examined a mechanism by which these cells, after phagocytic challenge, may control or amplify the inflammatory response in lung parenchyma. Normal human alveolar macrophages (AM) were studied from eight subjects. With in vitro culture, AM produced and released two substances into culture media which have potent chemoattractant activity for blood polymorphonuclear granulocytes (PMN) and negligible activity for mononuclear cells. Release of these factors is maximally stimulated by aggregated human immunoglobulin (Ig)G or zymosan particles; however, simple adhesion of the macrophages to plastic surfaces is also sufficient to stimulate release of these chemotactic substances. The larger substance (10,000 daltons) is immunologically distinct from C5a and interacts with a different PMN membrane receptor than that known to exist for formyl-methionyl-leucyl-phenylalanine. Its chemotactic activity is sensitive to the enzymatic effect of trypsin. Although producing a single elution peak on gelfiltration chromatography, electrofocusing in polyacrylamide gels yielded five peaks of radioactivity. Chemotactic activity was localized to a fraction with a pI = 5.0. The smaller molecular weight substance has been less well characterized. Thus, the human AM can produce at least two factors which attract PMN and this capability may augment the local inflammatory response in the lung.

Murine coronaviruses such as mouse hepatitis virus (MHV) infect mouse cells via cellular receptors that are isoforms of biliary glycoprotein (Bgp) of the carcinoembryonic antigen gene family (G. S. Dveksler, C. W. Dieffenbach, C. B. Cardellichio, K. McCuaig, M. N. Pensiero, G.-S. Jiang, N. Beauchemin, and K. V. Holmes, J. Virol. 67:1-8, 1993). The Bgp isoforms are generated through alternative splicing of the mouse Bgp1 gene that has two allelic forms called MHVR (or mmCGM1), expressed in MHV-susceptible mouse strains, and mmCGM2, expressed in SJL/J mice, which are resistant to MHV. We here report the cloning and characterization of a new Bgp-related gene designated Bgp2. The Bgp2 cDNA allowed the prediction of a 271-amino-acid glycoprotein with two immunoglobulin domains, a transmembrane, and a putative cytoplasmic tail. There is considerable divergence in the amino acid sequences of the N-terminal domains of the proteins coded by the Bgp1 gene from that of the Bgp2-encoded protein. RNase protection assays and RNA PCR showed that Bgp2 was expressed in BALB/c kidney, colon, and brain tissue, in SJL/J colon and liver tissue, in BALB/c and CD1 spleen tissue, in C3H macrophages, and in mouse rectal carcinoma CMT-93 cells. When Bgp2-transfected hamster cells were challenged with MHV-A59, MHV-JHM, or MHV-3, the Bgp2-encoded protein served as a functional MHV receptor, although with a lower efficiency than that of the MHVR glycoprotein. The Bgp2-mediated virus infection could not be inhibited by monoclonal antibody CC1 that is specific for the N-terminal domain of MHVR. Although CMT-93 cells express both MHVR and Bgp2, infection with the three strains of MHV was blocked by pretreatment with monoclonal antibody CC1, suggesting that MHVR was the only functional receptor in these cells. Thus, a novel murine Bgp gene has been identified that can be coexpressed in inbred mice with the Bgp1 glycoproteins and that can serve as a receptor for MHV strains when expressed in transfected hamster cells.

Although antibodies can prevent or modulate lentivirus infections in nonhuman primates, the biological functions of antibody responsible for such effects are not known. We sought to determine the role of antibody-dependent cell-mediated virus inhibition (ADCVI), an antibody function that inhibits virus yield from infected cells in the presence of Fc receptor-bearing effector cells, in preventing or controlling SIVmac251 infection in rhesus macaques (Macaca mulatta). Using CEMx174 cells infected with simian immunodeficiency virus mac251 (SIVmac251), both polyclonal and monoclonal anti-SIV antibodies were capable of potent virus inhibition in the presence of human peripheral blood mononuclear cell (PBMC) effector cells. In the absence of effector cells, virus inhibition was generally very poor. PBMCs from healthy rhesus macaques were also capable of mediating virus inhibition either against SIVmac251-infected CEMx174 cells or against infected, autologous rhesus target cells. We identified both CD14(+) cells and, to a lesser extent, CD8(+) cells as the effector cell population in the rhesus PBMCs. Finally, pooled, nonneutralizing SIV-antibody-positive serum, shown in a previous study to prevent infection of neonatal macaques after oral SIVmac251 challenge, had potent virus-inhibitory activity in the presence of effector cells; intact immunoglobulin G, rather than F(ab')(2), was required for such activity. This is the first demonstration of both humoral and cellular ADCVI functions in the macaque-SIV model. ADCVI activity in nonneutralizing serum that prevents SIV infection suggests that ADCVI may be a protective immune function. Finally, our data underscore the potential importance of Fc-Fc receptor interactions in mediating biological activities of antibody.

Two classes of molecules often released after the interaction of T lymphocytes, macrophages and antigen are B-cell maturation factors (BMF)1-3 and immune (gamma) interferon (IFN-gamma)4-7. BMFs directly induce the maturation of resting B lymphocytes to the state of active immunoglobulin secretion, while IFN-gamma is defined by the reduction of viral infectivity in vitro. However, interferons have been shown to have a variety of effects and they have also been reported both to increase and decrease B-cell differentiation in intact animals and complex cellular mixtures in vitro. Here we show that murine IFN-gamma produced by recombinant DNA technology shows similar biological effects to BMFs from two other sources. All three preparations induce immunoglobulin secretion by both normal resting murine splenic B cells and the comparable B-cell tumour line WEHI-279.1 (refs 1, 3). IFN-gamma and the other two BMFs are not identical, however, as anti-IFN-gamma antibodies block the effects on B cells of IFN-gamma, but not those of the other two lymphokines. IFN-gamma may be one of several molecules with a direct role in driving the maturation of resting B cells to active immunoglobulin secretion.

Anti-protective antigen (PA) immunoglobulin (Ig) G, toxin neutralization, and PA-specific IgG memory B cell responses were studied in patients with bioterrorism-related cutaneous or inhalation anthrax and in a patient with laboratory-acquired cutaneous anthrax. Responses were determined for >1 year after the onset of symptoms. Eleven days after the onset of symptoms (15 days after likely exposure), anti-PA IgG was detected in 16 of 17 patients with confirmed or suspected clinical anthrax who were tested. Anti-PA IgG remained detectable 8-16 months after the onset of symptoms in all 6 survivors of inhalation anthrax and in 7 of 11 survivors of cutaneous anthrax who were tested. Anti-PA IgG levels and serum toxin neutralizing activity were strongly associated (R2=0.83). PA-specific IgG memory B cells were detectable in all 6 survivors of inhalation anthrax but in only 2 of 7 patients with cutaneous anthrax who were tested. Anti-PA IgG is an important diagnostic marker of anthrax, a predictor of serum anti-toxin activity, and a marker of immunological memory against anthrax.

Antibody responses to salivary gland sonicate (SGS) from Lutzomyia longipalpis were investigated using serum samples from individuals living in an area where visceral leishmaniasis is endemic. Individuals were classified into 2 groups, according to the alteration of their responses to Leishmania chagasi antigen over the course of 6 months. Group 1 included children who experienced anti-L. chagasi seroconversion from negative to positive; group 2 included children who experienced delayed-type hypersensitivity (DTH) response to L. chagasi antigen conversion from negative to positive. Individuals who experienced seroconversion against L. chagasi antigens did not have increased anti-saliva antibody response, whereas those who developed a positive anti-L. chagasi DTH response had increased immunoglobulin (Ig) G, IgGGS antibody levels. Despite wide variation, serum samples from individuals in group 2 recognized more bands in SGS than did those from individuals in group 1. This simultaneous appearance of anti-saliva humoral response and anti-L. chagasi cell-mediated immunity supports the hypothesis that induction of immune response against SGS can facilitate induction of a protective response against leishmaniasis.

Burkholderia pseudomallei is the causative agent of melioidosis, an infectious disease with protean clinical manifestations. The major route of infection is thought to be through subcutaneous inoculation of contaminated soil and water, although ingestion and inhalation of contaminated aerosols are also possible. This study examines infection through the intranasal route in a murine model to mimic infection through inhalation. Two strains of mice, C57BL/6 and BALB/c, exhibit differential susceptibilities to the infection, with the C57BL/6 mice being considerably more resistant. To examine host factors that could contribute to this difference, bacterial loads and cytokine profiles in the two strains of mice were compared. We found that infected BALB/c mice exhibited higher bacterial loads in the lung and spleen and that they produced significantly higher levels of gamma interferon (IFN-gamma) in the serum than C57BL/6 mice. Although tumor necrosis factor alpha and interleukin-1 could be detected in the nasal washes and sera of both strains of mice, the production in serum was transient and much lower than that of IFN-gamma. C57BL/6 mice also exhibited memory responses to bacteria upon reinfection, with the production of serum immunoglobulin G (IgG) and mucosal IgA antibodies. Thus, it is possible that the production of systemic and mucosal antibodies is important for protection against disease in C57BL/6 mice.

The feasibility of using the highly purified native attachment (G) protein in a subunit vaccine against respiratory syncytial virus (RSV) was examined in a murine model with or without the fusion (F) protein of RSV and the adjuvant QS-21. The studies established that QS-21 was more potent than AIOH as an adjuvant for both F and G glycoproteins. Augmented antigen-dependent killer cell activity and complement-assisted serum neutralizing and anti-F and G protein immunoglobulin GG/QS-21 generated immune responses that were characterized by low levels of antigen-dependent killer cell activity, elevated levels of interleukin-5 (IL-5) and percentages of eosinophils in the bronchoalveolar lavage fluids after challenge, and splenic immunocytes that secreted IL-5 but not gamma interferon (IFN-gamma) after in vitro stimulation with purified whole virus antigens. The pulmonary eosinophilia was similar to that induced by a facsimile of a formalin-inactivated vaccine used in previous clinical trials and was prevented by prior in vivo treatment with anti-IL-5 but not with control immunoglobulin G or anti-IFN-gamma neutralizing monoclonal antibodies. Thus the data implied that vaccination with G/QS-21 generated helper T-cell immune responses that were type 2 in nature. Alternatively, the data suggested that the helper T-cell immune responses elicited by F/QS-21 were more type 1 in character. Neither eosinophilia nor elevated levels of IL-5 were observed in the lungs of mice after challenge. Noteworthy levels of antigen-dependent killer cell activity was observed, and splenic immunocytes secreted copious quantities of IFN-gamma. Immunization with a combination vaccine composed of highly purified native F and G proteins plus QS-21 (F+G/QS-21) resulted in augmented complement-assisted serum neutralizing antibody titers compared with vaccination with either F/QS-21 or G/QS-21 alone. However, following vaccination with F+G/QS-21, the bronchoalveolar lavage fluids contained significant increases in IL-5 and percentages of eosinophils after challenge, the spleen cells appeared to secrete less IFN-gamma after in vitro stimulation, and there was no evidence of increased numbers of antigen-dependent killer cell precursors. Taken together, the data imply that native G protein influences the nature of the immune responses elicited by F/QS-21. The results therefore suggest that G, not F, protein has more potential to bias the host for atypical pulmonary inflammatory responses.

We have characterized the natural immune responses to the 19-kDa domain of merozoite surface protein 1 in individuals from an area of western Kenya in which malaria is holoendemic. We used the three known natural variant forms of the yeast-expressed recombinant 19-kDa fragment that are referred to as the E-KNG, Q-KNG, and E-TSR antigens. T-cell proliferative responses in individuals older than 15 years and the profile of immunoglobulin G (IgG) antibody isotypes in individuals from 2 to 74 years old were determined. Positive proliferative responses to the Q-KNG antigen were observed for 54% of the individuals, and 37 and 35% of the individuals responded to the E-KNG and E-TSR constructs, respectively. Considerable heterogeneity in the T-cell proliferative responses to these three variant antigens was observed in different individuals, suggesting that the 19-kDa antigen may contain variant-specific T epitopes. Among responses of the different isotypes of the IgG antibody, IgGGGGGGG and Q-KNG constructs and not the E-TSR antigen. This result suggests that the fine specificity of IgGG and TSR) of the antigen.

Immunodeficient mice bearing components of a human immune system present a novel approach for studying human immune responses. We investigated the number, phenotype, developmental kinetics, and function of developing human immune cells following transfer of CD34(+) hematopoietic stem cell (HSC) preparations originating from second trimester human fetal liver (HFL), umbilical cord blood (UCB), or granulocyte colony-stimulating factor-mobilized adult blood (G-CSF-AB) delivered via intrahepatic injection into sublethally irradiated neonatal NOD-scid/gammac(-/-), Balb/c-Rag1(-/-)gammac(-/-), and C.B-17-scid/bg mice. HFL and UCB HSC provided the greatest number and breadth of developing cells. NOD-scid/gammac(-/-) and Balb/c-Rag1(-/-)gammac(-/-) harbored human B and dendritic cells as well as human platelets in peripheral blood, whereas NOD-scid/gammac(-/-) mice harbored higher levels of human T cells. NOD-scid/gammac(-/-) mice engrafted with HFL CD34(+) HSC demonstrated human immunological competence evidenced by white pulp expansion and increases in total human immunoglobulin following immunization with T-dependent antigens and delayed-type hypersensitivity-infiltrating leukocytes in response to antigenic challenge. In conclusion, we describe an encouraging base system for studying human hematopoietic lineage development and function utilizing human HFL or UCB HSC-engrafted NOD-scid/gammac(-/-) mice that is well suited for future studies toward the development of a fully competent humanized mouse model.