Isotype Switching Increases Efficacy of Antibody Protection against Cryptococcus neoformans Infection in Mice

The isotype and epitope specificities of antibodies both contribute to the efficacy of antibodies that mediate immunity to Cryptococcus neoformans, but the relationship between these properties is only partially understood. In this study, we analyzed the efficacy of protection of two sets of immunoglobulin G (IgG) isotype switch variants from two IgG3 monoclonal antibodies (MAbs) which are either not protective or disease enhancing, depending on the mouse model used. The two IgG3 MAbs 3E5 and 4H3 have different epitope specificities. Protection experiments were done with A/JCr mice infected intravenously with C. neoformans and administered with 3E5 IgG3 and its IgG1, IgG2a, and IgG2b switch variants. These experiments revealed that IgG1, IgG2b, and IgG2a were each more effective than IgG3. For 4H3 IgG3 and its IgG1 and IgG2b switch variants, the relative efficacy was IgG2b > IgG1 >> IgG3. The combination of 3E5 IgG3 and 4H3 IgG3 was more deleterious than either IgG3 alone. All IgG isotypes were opsonic for mouse bronchoalveolar cells, with the relative efficacy being IgG2b > IgG2a > IgG1 > IgG3. These results (i) confirm that a nonprotective IgG3 MAb can be converted to a protective MAb by isotype switching, (ii) indicate that the efficacy of protection of an IgG1 MAb can be increased by isotype switching to another subclass, (iii) show that protective and nonprotective IgG MAbs are opsonic, and (iv) provide additional evidence for the concept that the efficacy of the antibody response to C. neoformans is dependent on the type of MAb elicited.Cryptococcus neoformans is a fungus which is a frequent cause of life-threatening meningoencephalitis in patients with impaired immunity (22, 25). Cryptococcosis has been reported to occur in 6 to 8% of patients with AIDS (7). In immunocompromised individuals, C. neoformans infections are often incurable with conventional antifungal agents, and these patients frequently require lifelong therapy (45). The difficulties involved in the management of cryptococcosis in immunocompromised individuals have led to a reexamination of the potential of antibody-mediated immunity for prevention and therapy of cryptococcal infections. A polysaccharide-tetanus toxoid (TT) conjugate vaccine which is highly immunogenic and can elicit protective antibodies in mice has been made (3, 8, 9). In addition, several monoclonal antibodies (MAbs) have been shown to modify the course of infection in mice, and these may be useful in therapy of human infection (12, 14, 28, 42, 43).Cell-mediated immunity is generally acknowledged to provide important host defense against C. neoformans infection (4, 20, 26, 31, 42). In contrast, the role of antibody-mediated immunity in host resistance is less certain (2), but there is considerable evidence that administration of some MAbs can modify the course of infection in mice (8, 12, 14, 16, 28, 33). C. neoformans is unusual among fungal pathogens in that it has a polysaccharide capsule composed primarily of glucuronoxylomannan (GXM) (6), which is important for virulence (5). The capsular polysaccharide has been shown to produce a variety of deleterious effects including inhibition of phagocytosis (21), interference with antigen presentation (39), shedding of adhesion molecules (11), inhibition of leukocyte migration (10), and alterations in cytokine production by host effector cells (24, 40, 41). Antibodies to the C. neoformans capsular polysaccharide may contribute to host defense through multiple effects including enhanced opsonization (13, 18, 23, 30, 44), clearance of polysaccharide antigen (15), promotion of granuloma formation (14), and release of oxygen- and nitrogen-derived oxidants (27, 38).In previous studies, we demonstrated that immunoglobulin G3 (IgG3) MAbs are not protective in various mouse models of cryptococcal infection (32, 42). When one of these nonprotective IgG3 MAbs was switched to IgG1, the IgG1 significantly prolonged animal survival (32, 42). In the present study, we analyzed two families of IgG switch variants generated in vitro from two nonprotective IgG3 MAbs with different epitope specificities. We found that MAbs with different isotypes have different protective efficacies and that switching of nonprotective IgG3 MAbs to IgG1, IgG2b, and IgG2a significantly increased antibody protective efficacy. These studies demonstrate a complex relationship among efficacy of antibody protection, epitope specificity, and isotype.     

Adherence Patterns and Adherence-Related DNA Sequences in Escherichia coli Isolates from Children with and without Diarrhea in S?o Paulo City,Brazil

The correlation between various adherence patterns and adherence-related DNA sequences in Escherichia coli isolates from 1- to 4-year-old children with and without diarrhea in São Paulo, Brazil, was evaluated. A total of 1,801 isolates obtained from 200 patients and 200 age-matched controls were studied. The adherence patterns found were classified as diffuse, aggregative, aggregative in a 6-h assay, aggregative predominantly in coverslips, localized, localized-like, and noncharacteristic. In general, the DNA sequences used as probes showed excellent specificities (>93%), but their sensitivities varied. Thus, the results of bioassays and assays with DNA probes normally used to search for adherent E. coli did not correlate well, and the best method for the identification of these organisms in the clinical research setting remains controversial. Isolates presenting diffuse adherence or hybridizing with the related daaC probe, or both, were by far the most frequent in patients (31.5, 26.0, and 23.0%, respectively), followed by isolates presenting aggregative adherence or hybridizing with the related EAEC probe, or both (21.5, 13.0, and 10.5%, respectively). None of the different combinations of adherence patterns and adherence-related DNA sequences found were associated with acute diarrhea.The first step in the establishment of the diarrheal diseases caused by the various categories of diarrheagenic Escherichia coli is adherence to epithelial cells of the intestinal mucosa. In vitro assays with eukaryotic cell lines (HeLa and HEp-2 cells) have identified three distinct adherence patterns among fecal isolates of E. coli: localized, diffuse, and aggregative (37, 38, 41). Localized adherence (LA) is characterized by formation of bacterial microcolonies on a restricted area(s) of the cell surface, while diffuse adherence (DA) is the scattered attachment of bacteria over the whole surface of the cell (41). The pattern of aggregative adherence (AA) consists of bacterial attachment to the cells and the intervening cell growth surface in a stacked brick-like lattice (37).The LA pattern was first detected in strains classified as enteropathogenic E. coli (EPEC) among serogroups associated with outbreaks of infantile diarrhea (41). Although E. coli strains exhibiting DA (DAEC) have been isolated at similar frequencies from feces of infants and young children with acute diarrhea and nondiarrheic controls in some populations (3, 10, 11, 14, 18), they were significantly associated with diarrhea in other settings (1, 17, 24, 29, 33). E. coli strains showing AA, termed enteroaggregative E. coli (EAEC), have been linked to sporadic persistent diarrhea (3, 4, 7, 10, 13, 26, 27, 44) and to outbreaks of diarrhea in both developing and developed countries (8, 12, 28, 43). However, the role of EAEC in acute diarrhea is still controversial: some studies have shown a correlation (7, 23, 25, 27, 34, 37), but others (1, 3, 6, 10, 11, 13–15, 17, 18, 24, 26, 29, 33, 44) have not.DNA probes derived from adherence-related sequences have been constructed (2, 5, 16, 31, 36) and used in hybridization assays for the detection of the different established and putative categories of diarrheagenic E. coli in many epidemiological studies.We evaluated the relationship between the LA, DA, and AA patterns and hybridization with adherence-related DNA sequences and tested children 1 to 4 years old with and without acute diarrhea for the presence of adherent E. coli strains.     

d-Lactate Production and [14C]Succinic Acid Uptake by Adherent and Nonadherent Escherichia coli

Escherichia coli isolates of different adherence phenotypes produced different amounts of d-lactate. Alterations of culture conditions did not influence the amount of d-lactate produced. The observed pH decreases in tissue culture medium corresponded with increases in d-lactate concentration. Very little [¹⁴C]succinic acid was incorporated into cells during the in vitro incubation of adherent and nonadherent E. coli with HeLa cells, but the amounts of tracer removed from the culture medium by adherent and nonadherent strains differed. The results are further evidence of a difference in the metabolic behavior of adherent and nonadherent E. coli.One of the virulence associated properties of enteropathogenic Escherichia coli (5, 13, 14) is the ability to adhere to small intestinal mucosa (3, 11, 12, 21, 24, 26, 27). Although this adherence is an important event in the induction of diarrhea, the mechanism by which adherent E. coli mediates pathogenicity remains uncertain (1, 2, 7, 18, 26, 27).Several studies have shown that the in vitro adherence of E. coli to HEp-2 or HeLa cells in tissue culture can be used as a marker of enteroadherence (4, 6, 8, 9, 15, 16, 19, 22, 23, 28, 29). We used the HeLa assay (20) to detect this virulence characteristic in E. coli isolates from infants with acute diarrhea and, during the 3-h assay, observed E. coli-induced changes in the pH of the tissue culture medium (17). The pH changes induced by organisms with different adherence phenotypes differed. Since the characteristic end products of E. coli fermentation include lactic acid, succinic acid, and acetic acid, the pH changes could be explained by differences in the production of organic acids. Other plausible explanations are differences in the removal of organic acids from the medium and interactions between bacteria and HeLa cells during adherence.This paper describes two sets of experiments, one based on the production of lactic acid and the other on the removal of succinic acid from the medium. The objectives were to determine (i) whether there is a metabolic difference between localized, diffuse, and nonadherent isolates in the amount of lactate produced or succinate removed from the incubation medium, (ii) whether E. coli changes from aerobic to anaerobic metabolism during incubation periods of up to 5 h under different culture conditions, (iii) whether an increase in lactate production or succinate removal coincides with the drop in pH previously observed, and (iv) whether the pH changes can be attributed to differences in bacterial growth rates between isolates with different in vitro adherence patterns and nonadherent strains.     

Bivalency Is Required for Anticapsular Monoclonal Antibodies To Optimally Suppress Activation of the Alternative Complement Pathway by the Cryptococcus neoformans Capsule

Encapsulated cells of Cryptococcus neoformans are potent activators of the alternative complement pathway. Previous studies found that monoclonal antibodies (MAbs) specific for the major capsular polysaccharide, termed glucuronoxylomannan (GXM), can markedly suppress the ability of the capsule to accumulate C3 from normal human serum via the alternative pathway. The present study examined the abilities of F(ab)₂ and Fab fragments of three MAbs (MAbs 439, 3C2, and 471) to mediate the suppressive effect. The results showed that F(ab)₂ fragments of all three MAbs suppressed activation and binding of C3 via the alternative pathway in a manner similar to that of intact antibodies. In contrast, Fab fragments of MAb 439 and MAb 3C2 showed no suppressive activity, and Fab fragments of MAb 471 were markedly reduced in suppressive activity. Indeed, there was an earlier accumulation of C3 on encapsulated cryptococci in the presence of the Fab fragments. Study of subclass switch families of MAb 439 and MAb 471 found that MAbs of an immunoglobulin G (IgG) subclass with increased flexibility in the hinge region (IgG2b) had less suppressive activity than MAbs of IgG subclasses with less flexibility (IgG1 or IgG2a). Taken together, these results indicate that cross-linking of the capsular matrix is an essential component in suppression of the alternative complement pathway by anti-GXM MAbs.The capsule of the pathogenic yeast Cryptococcus neoformans is a powerful activator of the alternative complement pathway (8, 16). Incubation of encapsulated cryptococci in normal human serum (NHS) leads to deposition of 10⁷ to 10⁸ molecules of C3 onto the typical yeast cell (18, 38); the capsule itself is the site for C3 binding (19, 21). Such activation and binding of C3 is due solely to the action of the alternative pathway (20, 21, 37). Binding of C3 via the alternative pathway in NHS is characterized by a delay of approximately 4 to 6 min before bound C3 is readily detectable (20).The major component of the cryptococcal capsule is the high-molecular-weight polysaccharide glucuronoxylomannan (GXM). Several anti-GXM monoclonal antibodies (MAbs) have been shown to provide a measure of protection in a murine model of cryptococcosis (9, 24, 30). In the accompanying report, we have examined the ability of anti-GXM MAbs to initiate the classical pathway, leading to accelerated deposition of C3 onto the yeast (17). These studies showed that most anti-GXM MAbs promote early deposition of C3 fragments into the capsule. However, depending on the epitope specificity of the MAb, some anti-GXM MAbs markedly reduced the apparent rate of amplification of bound C3, with the net result that fewer C3 molecules bound to the cell over a 20- to 30-min incubation period than would have bound in the absence of the antibody. When classical pathway initiation was blocked by the use of EGTA to chelate Ca²⁺ (12, 28), antibodies with the suppressive epitope specificity almost completely blocked the normal alternative pathway activation and binding of C3 that would have occurred in the absence of the MAbs.The ability of an antibody to block antibody-independent activation of the alternative pathway is without an obvious parallel in the literature. There are several potential mechanisms for antibody-mediated suppression. First, the antibody could bind to and occlude specific sites on the capsule that might be preferred acceptors for metastable C3b. Second, the capsule could contain specific domains that regulate the ability of the capsule to activate the alternative pathway. Antibodies specific for such regulatory domains could influence the ability of the cell to activate the alternative pathway. Finally, multivalent antibody could cross-link the capsule in a manner that prevents effective amplification. For example, binding of a multivalent antibody could reduce the ability of metastable C3b to diffuse from sites of C3 convertase activity. We have reasoned that the first two mechanisms for antibody-induced suppression of C3 binding would be mediated by intact antibody, F(ab)₂ fragments of the antibody, and Fab fragments. In contrast, inhibition that is dependent on cross-linking of the capsular matrix would be mediated by intact antibodies and F(ab)₂ fragments but not by Fab fragments.The objective of our study was to examine three anticapsular MAbs that suppress alternative pathway-dependent C3 binding. The suppressive activities of intact antibodies, F(ab)₂ fragments, and Fab fragments were compared. The results showed that intact antibodies and F(ab)₂ fragments of the antibodies suppressed accumulation of C3 fragments on the capsule. In contrast, Fab fragments of the suppressive antibodies showed markedly reduced or no ability to block alternative pathway activation by the capsule; indeed, Fab fragments derived from suppressive antibodies accelerated activation and binding of C3 via the alternative pathway.     

Development of Loop-Mediated Isothermal Amplification Assay for Detection of Entamoeba histolytica

A novel one-step, closed-tube, loop-mediated isothermal amplification (LAMP) assay for detecting Entamoeba histolytica, one of the leading causes of morbidity in developing countries, was developed. The sensitivity of the LAMP assay is 1 parasite per reaction. A total of 130 clinical samples were analyzed, and the results compared with those of conventional nested PCR to validate the practicability of this assay. No DNA was amplified from other diarrheal pathogens, such as other Entamoeba species, bacteria, and viruses. These results indicate that LAMP is a rapid, simple, and valuable diagnostic tool for epidemiological studies of amebiasis.Entamoeba histolytica is the etiologic agent of human amebiasis, which causes an estimated 40 to 50 million cases of dysentery and liver abscess and up to 100,000 deaths each year, mainly in tropical and subtropical countries (6). The detection of amebiasis in developing countries is currently dependent on microscopic examination of stool samples from patients. However, this system is at best only 10 to 60% sensitive (9, 10, 14, 22) and is incapable of distinguishing between the pathogenic species E. histolytica, the nonpathogenic species Entamoeba dispar, and the amphizoic amoeba Entamoeba moshkovskii, which infects humans sporadically (5, 7, 8). The lack of early and accurate diagnosis is a critical obstacle to clinical management of E. histolytica infection globally. Therefore, an accurate but feasible method that can be easily used in areas where amebiasis is endemic is clearly needed to confirm the identification of E. histolytica.Several new diagnostic approaches based on the detection of an E. histolytica-specific antigen and DNA had been established recently (22). The TechLab Entamoeba histolytica II kit is the only commercially available antigen test kit for specific detection of E. histolytica in feces. However, since storage or the use of preservatives destroys the antigen, it is recommended for use exclusively with fresh stool samples (21, 22). Several PCR-based assays for E. histolytica identification have subsequently been published, including single-tube multiplex reactions and nested PCR (12). The results suggest that PCR should be useful as a reference test for sensitive differentiation of E. histolytica and E. dispar (7) and could replace the TechLab enzyme-linked immunosorbent assay in areas where the pathogen occurs less frequently (23). However, the PCR assay commonly requires electrophoresis to detect the amplicons, leading to relatively high expense, including labor costs, and long turnaround time. Real-time PCR assays have also been applied to detect and quantify pathogens by continuously monitoring the amplicon formation with time (22). Nevertheless, such assays require an expensive thermal cycler with a fluorescence detector.The loop-mediated isothermal amplification (LAMP) assay was originally developed by Mori et al. (17), Nagamine et al. (18), and Notomi et al. (19) (Eiken Chemical Co., Ltd., Japan), using a set of two specifically designed inner primers and two outer primers that recognize six distinct regions of the targeted DNA. Since this reaction is performed under isothermal conditions, simple incubators, such as a water bath or heat block, are adequate for the DNA amplification. Considering these advantages, the LAMP assay could become a valuable diagnostic tool in developing countries or hospital laboratories. Therefore, the aim of this study was to develop a sensitive and specific LAMP-based method to detect E. histolytica and to evaluate this method by comparing it to a conventional nested PCR assay with clinical fecal samples.     

Mannose Induces the Release of Cytopathic Factors from Acanthamoeba castellanii

Acanthamoeba keratitis is a chronic inflammatory disease of the cornea which is highly resistant to many antimicrobial agents. The pathogenic mechanisms of this disease are poorly understood. However, it is believed that the initial phases in the pathogenesis of Acanthamoeba keratitis involve parasite binding and lysis of the corneal epithelium. These processes were examined in vitro, using Acanthamoeba castellanii trophozoites. Parasites readily adhered to Chinese hamster corneal epithelial cells in vitro; however, parasite binding was strongly inhibited by mannose but not by lactose. Although mannose prevented trophozoite binding, it did not affect cytolysis of corneal epithelial cells. Moreover, mannose treatment induced trophozoites to release cytolytic factors that lysed corneal epithelial cells in vitro. These factors were uniquely induced by mannose because supernatants collected from either untreated trophozoites or trophozoites treated with other sugars failed to lyse corneal cells. The soluble factors were size fractionated in centrifugal concentrators and found to be ≥100 kDa. Treatment of the supernatants with the serine protease inhibitor phenylmethylsulfonyl fluoride inhibited most, but not all, of the cytopathic activity. These data suggest that the binding of Acanthamoeba to mannosylated proteins on the corneal epithelium may exacerbate the pathogenic cascade by initiating the release of cytolytic factors.Acanthamoeba spp. are protozoal parasites capable of infecting the skin, brain, and eye (10, 15, 17, 31, 32, 37). Corneal inflammation produced by Acanthamoeba was first recognized in 1973 and has since been intimately associated with contact lens wear (15, 31). Often the disease displays a ring-like neutrophilic stromal infiltrate with an overlying epithelial ulcer. The epithelium often undergoes a recurrent cycle of healing and breakdown during the progression of the disease. Topical or systemic treatment with antibiotics, antifungals, corticosteroids, and antivirals is often ineffectual (2). Typical treatment consists of around-the-clock hourly topical treatments with propamidine isothionate, polyhexamethylene biguanide, neomycin, or chlorhexidine, alone or in combination. This therapeutic regimen may continue for weeks. Many patients receive therapeutic corneal transplants, which can be reinfected by quiescent parasites residing in the periphery of the cornea.Parasite binding to the corneal epithelium is believed to be an important first step in the infectious cascade of Acanthamoeba keratitis. We have shown that adherence of Acanthamoeba to corneal buttons in vitro varies among mammalian species and correlates with susceptibility to experimental Acanthamoeba keratitis (14, 19, 35). Parasitic infections, such as Acanthamoeba keratitis, often occur in a sequential manner and are initiated by the pathogen’s adherence to host cells. Bacteria, fungi, and amoebae have been shown to bind to epithelial cells via lectin-glycoprotein interactions (5, 6, 11, 18, 20–22, 26, 27, 40). The cell surface of Pseudomonas aeruginosa is decorated with lectins which bind surface glycoproteins of the epithelium to be invaded (30, 39). Entamoeba histolytica also utilizes glycoproteins as receptor ligands for adherence to the gastrointestinal epithelium (6, 16, 25–29). Binding of Acanthamoeba polyphaga and A. castellanii to corneal epithelial cells in culture is inhibited by mannose (18, 40). Subsequent studies have indicated that the binding of A. castellanii to corneal epithelial cells is mediated by a 136-kDa mannose-binding protein on the trophozoite cell membrane (40).The pathophysiology of Acanthamoeba keratitis is poorly understood. Several studies have demonstrated that Acanthamoeba trophozoites can induce either cytolysis or apoptosis of target cells in vitro (1, 7, 24, 33, 34). Pathogenic Acanthamoeba trophozoites produce a variety of proteases which are believed to facilitate parasite penetration into the corneal stroma (9). Once in the stroma, Acanthamoeba trophozoites secrete collagenolytic enzymes which contribute to the dissolution of the stromal matrix (13).This study was undertaken to examine the cytopathic mechanisms employed by Acanthamoeba during the initial phase of ocular infection. We tested the hypothesis that blocking parasite binding to corneal epithelial cells with mannose would prevent parasite-mediated cytolysis and invasion of the corneal stroma. The results, however, indicate that although mannose blocks parasite binding, it also facilitates the release of cytolytic factors which kill corneal epithelial cells.     

Characterization of Anticapsular Monoclonal Antibodies That Regulate Activation of the Complement System by the Cryptococcus neoformans Capsule

Incubation of the encapsulated yeast Cryptococcus neoformans in human serum leads to alternative pathway-mediated deposition of C3 fragments in the capsule. We examined the ability of monoclonal antibodies (MAbs) specific for different epitopes of the major capsular polysaccharide to alter the kinetics for classical and alternative pathway-mediated deposition of C3 onto a serotype A strain. We studied MAbs reactive with capsular serotypes A, B, C, and D (MAb group II); serotypes A, B, and D (MAb group III); and serotypes A and D (MAb group IV). The MAb groupings are based on antibody variable region usage which determines the antibody molecular structure. When both the classical and alternative pathways were operative, group II MAbs induced early classical pathway-mediated binding of C3 but reduced the overall rate of C3 accumulation and the amount of bound C3. Group III MAbs closely mimicked the effects of group II MAbs but exhibited reduced support of early classical pathway-facilitated accumulation of C3. Depending on the antibody isotype, group IV MAbs slightly or markedly enhanced early binding of C3 but had no effect on either the rate of C3 accumulation or the amount of bound C3. When the classical pathway was blocked, group II and III MAbs markedly suppressed C3 binding that normally would have occurred via the alternative pathway. In contrast, MAbs of group IV had no effect on alternative pathway-mediated C3 binding. These results indicate that anticapsular antibodies with different epitope specificities may have distinct regulatory effects on activation and binding of C3.Cryptococcus neoformans is the etiological agent of cryptococcal meningitis, a life-threatening infection of particular importance in patients with deficiencies in cellular immunity, most notably patients with the AIDS. The yeast is surrounded by a polysaccharide capsule that is composed primarily of glucuronoxylomannan (GXM), which has a linear (1→3)-α-d-mannopyranan backbone bearing β-d-xylopyranosyl, β-d-glucopyranosyluronic acid, and O-acetyl substituents (3, 9, 54). The cryptococcal capsule occurs as four major serotypes (A, B, C, and D) and is an essential virulence factor for the yeast.One of the most striking features of the cryptococcal capsule is its ability to activate the alternative complement pathway. Incubation of encapsulated cryptococci in normal human serum (NHS) leads to the deposition of 10⁷ to 10⁸ C3 fragments on the yeast (28, 56). The C3 is deposited at the surface and throughout the capsule (30). Available evidence indicates that the amount of anti-GXM antibodies found in NHS is not sufficient to initiate the classical pathway (24); consequently, activation and binding of C3 to the cryptococcal capsule are mediated entirely by the alternative complement pathway (29, 30, 55). One of the hallmark features of alternative pathway deposition of C3 onto encapsulated cryptococci is a delay of 5 to 8 min before readily detectable amounts of C3 are found on yeast cells incubated in NHS (29, 55). Once past the initial lag, C3 fragments rapidly accumulate on the yeast cells as incubation proceeds for an additional 10 min.Recently, there has been interest in antibody-mediated resistance to cryptococcosis. Monoclonal antibodies (MAbs) have been proposed for treatment of cryptococcosis (7), and immunization with GXM-protein conjugates has been suggested for prevention of cryptococcosis (6, 12, 13). However, it is becoming increasingly clear that anti-GXM MAbs may have distinct specificities and biological activities. Anti-GXM MAbs which differ in (i) reactivities with GXM of the four major serotypes (2), (ii) apparent binding sites in the cryptococcal capsule (32, 37), and (iii) abilities to provide protection in a murine model of cryptococcosis (32, 37) have been described. Some differences in biological activity are related to differences in the epitope specificities of the various MAbs (32, 37).One means by which antibodies could enhance resistance to cryptococcosis is through accelerated deposition of opsonic C3 fragments via the action of the classical pathway. Such an acceleration would reduce or eliminate the 5- to 8-min lag that occurs during alternative pathway-mediated deposition of C3 fragments. The objectives of our study were to evaluate the effects of anti-GXM MAbs on the kinetics and sites for deposition of C3 fragments into the cryptococcal capsule. We examined several well-characterized antibodies that differed in the epitope specificity of the MAbs. The results showed that MAbs with different isotypes and epitope specificities had distinctly different effects on activation and binding of C3 via the classical and alternative pathways; many antibodies markedly suppressed C3 binding, some antibodies accelerated C3 binding, and other antibodies had little or no effect.     

Immunodominant Major Outer Membrane Proteins of Ehrlichia chaffeensis Are Encoded by a Polymorphic Multigene Family

Several immunodominant major proteins ranging from 23 to 30 kDa were identified in the outer membrane fractions of Ehrlichia chaffeensis and Ehrlichia canis. The N-terminal amino acid sequence of a 28-kDa protein of E. chaffeensis (one of the major proteins) was determined. The gene (p28), almost full length, encoding the 28-kDa protein was cloned by PCR with primers designed based on the N-terminal sequence of the E. chaffeensis 28-kDa protein and the consensus sequence between the C termini of the Cowdria ruminantium MAP-1 and Anaplasma marginale MSP-4 proteins. The p28 gene was overexpressed, and antibody to the recombinant protein was raised in a rabbit. The antibody and serum from a patient infected with E. chaffeensis reacted with the recombinant protein, three proteins (29, 28, and 25 kDa) of E. chaffeensis, and a 30-kDa protein of E. canis. Immunoelectron microscopy with the rabbit antibody revealed that the antigenic epitope of the 28-kDa protein was exposed on the surface of E. chaffeensis. Southern blot analysis with a ³²P-labeled p28 gene probe revealed multiple copies of genes homologous to p28 in the E. chaffeensis genome. Six copies of the p28 gene were cloned and sequenced from the genomic DNA by using the same probe. The open reading frames of these gene copies were tandemly arranged with intergenic spaces. They were nonidentical genes and contained a semivariable region and three hypervariable regions in the predicted protein molecules. One of the gene copies encoded a protein with an internal amino acid sequence identical to the chemically determined N-terminal amino acid sequence of a 23-kDa protein of E. chaffeensis. Immunization with the recombinant P28 protein protected mice from infection with E. chaffeensis. These findings suggest that the 30-kDa-range proteins of E. chaffeensis represent a family of antigenically related homologous proteins encoded by a single gene family.

Ehrlichia chaffeensis, which causes human monocytic ehrlichiosis, is an obligate intracellular bacterium of monocytes and macrophages and belongs to the family Rickettsiaceae. Human ehrlichiosis is a tick-borne illness and was first reported in 1987 in the United States (21). Most patients have fever, chills, headache, arthralgia, myalgia, and hematologic abnormalities, including thrombocytopenia and leukopenia. Elevation of liver enzymes occurs in most patients. Since 1987, over 400 cases of human ehrlichiosis, detected primarily by serological means, have been reported in 30 states (3, 14, 16).Recently, several protein antigens of E. chaffeensis were identified by Western blot analysis with naturally infected human sera, experimentally inoculated dog sera, or monoclonal antibodies (7–10, 13, 30, 35, 40–42). Two of these antigens, namely, a heat shock protein (HSP) 60 homolog (35) and a 120-kDa protein (41, 42), have been cloned, sequenced, and expressed. Two E. chaffeensis proteins ranging from 28 to 30 kDa were shown to be dominant antigens and were cross-reactive between two Ehrlichia spp.: E. chaffeensis and E. canis (7, 30). Studies with monoclonal antibodies (MAbs) against E. chaffeensis showed that two or three proteins of from 22 to 30 kDa react with three MAbs by Western blotting and that these antigens are exposed on the surface of the organism as determined by immunogold labeling of negatively staining ehrlichiae (8–10, 40). However, why multiple proteins of different molecular sizes react with the MAbs has not been answered. These E. chaffeensis antigens in the 30-kDa range have not been examined at the molecular level.In this study, we demonstrated that a potentially immunoprotective 28-kDa protein (designated P28) located on the E. chaffeensis surface and antigenically cross-reactive proteins in the 30-kDa range are encoded by a multigene family.     

Characterization of the Roles of Hemolysin and Other Toxins in Enteropathy Caused by Alpha-Hemolytic Escherichia coli Linked to Human Diarrhea

Escherichia coli strains producing alpha-hemolysin have been associated with diarrhea in several studies, but it has not been clearly demonstrated that these strains are enteropathogens or that alpha-hemolysin is an enteric virulence factor. Such strains are generally regarded as avirulent commensals. We examined a collection of diarrhea-associated hemolytic E. coli (DHEC) strains for virulence factors. No strain produced classic enterotoxins, but they all produced an alpha-hemolysin that was indistinguishable from that of uropathogenic E. coli strains. DHEC strains also produced other toxins including cytotoxic necrotizing factor 1 (CNF1) and novel toxins, including a cell-detaching cytotoxin and a toxin that causes HeLa cell elongation. DHEC strains were enteropathogenic in the RITARD (reversible intestinal tie adult rabbit diarrhea) model of diarrhea, causing characteristic enteropathies, including inflammation, necrosis, and colonic cell hyperplasia in both small and large intestines. Alpha-hemolysin appeared to be a major virulence factor in this model since it conferred virulence to nonpathogenic E. coli strains. Other virulence factors also appear to be contributing to virulence. These findings support the epidemiologic link to diarrhea and suggest that further research into the role of DHEC and alpha-hemolysin in enteric disease is warranted.Escherichia coli is one of the major causes of human infectious diseases, partly because of the wide variety of virulence mechanisms and pathotypes (15), and new pathotypes continue to be described. A new pathotype was proposed by Gunzburg et al. after examining diarrheal pathogens in a prospective community-based study among Australian Aboriginal children (22). One group of isolates was significantly (P < 0.05) associated with diarrhea, and these isolates were particularly common among children younger than 18 months. The isolates did not produce any recognized enterotoxin or classic enteric virulence factor, although they exhibited diffuse or aggregative adhesion in a modified adhesion assay (15). All isolates were able to detach HEp-2 cell monolayers and were termed “cell-detaching E. coli.” This property was shown to be mediated by alpha-hemolysin, and we demonstrate below that all cell-detaching E. coli strains produce alpha-hemolysin and that some may also produce cytotoxic necrotizing factor 1 (CNF1) and other toxins. However, neither alpha-hemolysin nor CNF1 has been clearly demonstrated to be an enteric virulence factor, and the role of hemolysin in particular is controversial. We will refer to these isolates as diarrhea-associated hemolytic E. coli (DHEC) isolates.Alpha-hemolytic E. coli strains have been associated with human enteric disease, especially among young children (8, 10–12, 20–22), and the related enterohemolysin of E. coli O157 (35) appears to be involved in enteric disease. There has, however, been no large prospective case-controlled epidemiologic study of the association of alpha-hemolysin with human diarrhea. Alpha-hemolytic bacteria are also associated with enteric disease and diarrhea in pigs, cattle, and dogs (9, 13, 33, 36, 44, 45). Porcine diarrheal strains are almost universally hemolytic (23a), and alpha-hemolysin in these isolates enhanced virulence and colonization (37) but was not itself diarrheagenic. More recent studies have found that Hly⁺ CNF1⁺ strains caused fluid accumulation in piglets (33) and that neonatal pigs were susceptible to challenge with Hly⁺ CNF⁺ strains, which caused bloody diarrhea, enterocolitis, and systemic disease (45).In contrast, some earlier studies were unable to demonstrate a role for hemolysin in enteric disease, since neither hemolytic bacteria nor their supernatants caused fluid accumulation in ileal loops (10, 14, 37). Hemolytic strains may be isolated from the feces of asymptomatic people (26), and, among humans, hemolysin is more commonly associated with strains causing extraintestinal infections (5, 26).The genetics and in vitro mechanisms of alpha-hemolysin are well known. The hlyCABD operon encodes the structural 110-kDa hemolysin protein (HlyA) and proteins involved in processing and export (42). Once secreted, hemolytic activity is short-lived, and this has complicated studies of hemolysin toxigenicity (42). Hemolysin does not require a receptor to bind to target cells, inserting instead into the target cell membrane to form a pore that allows the free flow of cations, sugars, and water. This leads to leakage of intracellular contents and affects the cytoskeleton and metabolism (4, 9, 42, 43). In extraintestinal infections, hemolysin has multiple effects and roles, including resistance to host defense, tissue damage, and lethality, either by direct action or by stimulation of inflammatory mediators and signal transduction pathways (7, 9, 16, 42).CNF is a 114-kDa protein with homology to a family of dermonecrotic toxins (18) and is encoded by the monocistronic cnf gene, which lies just downstream of hly. The CNF1 toxin causes HeLa cells to become large and multinucleated as a result of actin disassembly, which results from activation of Rho (10, 19, 31). Similar to alpha-hemolysin, the role of CNF1 in diarrhea remains unclear. CNF1-producing strains have been isolated from diarrheal stools and have been associated with several outbreaks in humans (8, 10) and animals (13, 33, 44). Unfortunately, no large, prospective, case-controlled studies have been performed, and the best evidence for the pathogenicity of CNF1-toxigenic isolates is the marked virulence in piglet challenge experiments (45), outlined above. Purified CNF1 did not show enterotoxic potential in the suckling mouse or induce fluid accumulation in the rabbit ileal loop (10, 14), in contrast to the related CNF2, which is linked to enteric disease in animals (13, 14, 30). Both CNF toxins are extremely lethal, and have a variety of in vivo effects including tissue necrosis and edema (12–14).In this paper, we characterize DHEC isolates that were obtained from a study where alpha-hemolysin was significantly associated with disease (22) and show that they are able to cause disease in rabbits. Using molecular genetics, we attempt to analyze the role of each gene in pathogenesis.     

Molecular Cloning and Sequencing of Three Granulocytic Ehrlichia Genes Encoding High-Molecular-Weight Immunoreactive Proteins

Granulocytic Ehrlichia was isolated from canine blood obtained from animals challenged with field-collected Ixodes scapularis and propagated in HL60 cells. PCR primers specific for the 16S ribosomal DNA (rDNA) of the Ehrlichia genogroup comprising E. equi, E. phagocytophila, and the agent of human granulocytic ehrlichiosis (HGE) amplified DNA from extracts of these cells. Sequence analysis of this amplified DNA revealed that it is identical to the 16S rDNA sequence of the HGE agent. A genomic library was constructed with DNA from granulocytic Ehrlichia and screened with pooled sera from tick-challenged, granulocytic Ehrlichia-infected dogs. Several clones were isolated and sequenced. Three complete genes encoding proteins with apparent molecular masses of 100, 130, and 160 kDa were found. The recombinant proteins reacted with convalescent-phase sera from dogs and human patients recovering from HGE. This approach will be useful for identifying candidate diagnostic and vaccine antigens for granulocytic ehrlichiosis and aid in the classification of genogroup members.Members of the genus Ehrlichia include species which have a tropism for mononuclear phagocytes (E. canis, E. chaffeensis, E. muris, E. sennetsu, and E. risticii) (33) and those which infect granulocytes (E. ewingii [33], E. phagocytophila [10, 33], E. equi [14, 26], and the recently discovered agent of human granulocytic ehrlichiosis [HGE] [3, 7]). Disease caused by granulocytic Ehrlichia (GE) is manifested by fever, lethargy, thrombocytopenia, and death, and many species from diverse geographical locations have shown evidence of natural infection, including horses (25, 27, 29, 38), dogs (24, 34, 35), small mammals (39, 40), and humans (4, 15).The similar host range and near identity of the 16S rRNA genes of E. phagocytophila, E. equi, and the HGE agent (7) have raised the possibility that these organisms represent a single species (2, 28). In addition, Dumler et al. (9) have shown that they share significant antigenicity by immunofluorescence and immunoblot assays. Objective methods for species classification, e.g., molecular genetic analysis, have not been readily available, primarily because of an inability to culture these ehrlichiae in vitro. However, we (reference 43 and unpublished data) and others (13) have recently demonstrated successful cultivation of GE isolates from dogs and humans, respectively.In this paper, we describe the use of purified GE obtained from in vitro culture of infected HL60 cells, a promyelocytic human cell line, to generate a genomic DNA library for expression screening with sera obtained from dogs experimentally infected with GE. The screening resulted in the isolation of recombinant clones containing complete genes encoding three putative proteins of GE, GE 160, GE 130, and GE 100 (named for apparent molecular mass in kilodaltons). One of these proteins, the 100-kDa protein, is similar in both glutamic acid content and repeated amino acid structure to an immunodominant 120-kDa E. chaffeensis protein (45). Both the 100- and 130-kDa granulocytic Ehrlichia proteins share some amino acid sequence homology to the 120-kDa E. chaffeensis protein.     

In Vivo Regulation of Replicative Legionella pneumophila Lung Infection by Endogenous Interleukin-12

The in vivo role of endogenous interleukin 12 (IL-12) in modulating intrapulmonary growth of Legionella pneumophila was assessed by using a murine model of replicative L. pneumophila lung infection. Intratracheal inoculation of A/J mice with virulent bacteria (10⁶ L. pneumophila cells per mouse) resulted in induction of IL-12, which preceded clearance of the bacteria from the lung. Inhibition of endogenous IL-12 activity, via administration of IL-12 neutralizing antiserum, resulted in enhanced intrapulmonary growth of the bacteria within 5 days postinfection (compared to untreated L. pneumophila-infected mice). Because IL-12 has previously been shown to modulate the expression of cytokines, including gamma interferon (IFN-γ), tumor necrosis factor alpha (TNF-α), and IL-10, which regulate L. pneumophila growth, immunomodulatory effects of endogenous IL-12 on intrapulmonary levels of these cytokines during replicative L. pneumophila lung infection were subsequently assessed. Results of these experiments demonstrated that TNF-α activity was significantly lower, while protein levels of IFN-γ and IL-10 in the lung were similar, in L. pneumophila-infected mice administered IL-12 antiserum, compared to similarly infected untreated mice. Together, these results demonstrate that IL-12 is critical for resolution of replicative L. pneumophila lung infection and suggest that regulation of intrapulmonary growth of L. pneumophila by endogenous IL-12 is mediated, at least in part, by TNF-α.

Legionella pneumophila, the causative agent of Legionnaires’ disease, is an intracellular pathogen of mononuclear phagocytic cells (MPCs) (37, 43, 45). Pulmonary infection usually develops following inhalation of L. pneumophila-contaminated water aerosols or microaspiration of contaminated water sources (9). Following inhalation, the bacteria invade and replicate in host MPCs, primarily in alveolar MPCs (34, 36, 37, 43, 45). Intracellular growth of L. pneumophila results in eventual lysis of infected MPCs, the release of bacterial progeny, and reinfection of additional pulmonary cells (34, 36). Severe lung damage, mediated by tissue-destructive substances likely derived from both damaged host cells and the bacteria, ensues (20, 21).Previous studies have demonstrated that resistance to primary replicative L. pneumophila lung infection is dependent on the induction of cellular immunity and is mediated in part by cytokines including gamma interferon (IFN-γ) and tumor necrosis factor alpha (TNF-α) (8, 12, 14, 15, 23, 27, 28, 35, 57). Growth of L. pneumophila within permissive MPCs requires iron. IFN-γ limits MPC iron, thereby converting the MPC intracellular environment from one that is permissive to one that is nonpermissive for L. pneumophila replication (14, 15). IFN-γ in combination with other cytokines including TNF-α facilitates elimination of L. pneumophila from infected MPCs, likely through the induction of effector molecules including nitric oxide (12). In contrast, other cytokines including interleukin 10 (IL-10) facilitate growth of L. pneumophila in permissive MPCs, due in part to IL-10-mediated inhibition of TNF-α secretion and IFN-γ-mediated MPC activation (46).IL-12 is a recently described cytokine with pleiotropic effects on T cells and natural killer (NK) cells which include (i) regulation of expression of cytokines including IFN-γ, TNF-α, and IL-10 by T cells and/or NK cells, (ii) induction of T-cell and/or NK cell proliferation and/or differentiation, and (iii) enhancement of NK cell and T-cell cytotoxic activity (4, 5, 19, 32, 33, 39, 44, 47, 48, 50, 56). While systemic administration of exogenous IL-12 has been demonstrated to increase host resistance to several intracellular pathogens, including Leishmania major, Toxoplasma gondii, Listeria monocytogenes, Mycobacterium tuberculosis, Mycobacterium avium, and Plasmodium chabaudi, in mice (26, 29, 33, 40, 51, 52, 55), the role of endogenous IL-12 in innate immunity to intracellular pathogens including L. pneumophila has not been thoroughly investigated. We have recently developed a model of replicative L. pneumophila lung infection in A/J mice inoculated intratracheally with virulent bacteria and have used this model system to identify immune responses which mediate host resistance to legionellosis (10–12). Using this murine model of Legionnaires’ disease, we assessed the biologic relevance and immunomodulatory role of endogenous IL-12 in innate immunity to replicative L. pneumophila lung infection.     

Evidence of Nosocomial Infection in Japan Caused by High-Level Gentamicin-Resistant Enterococcus faecalis and Identification of the Pheromone-Responsive Conjugative Plasmid Encoding Gentamicin Resistance

A total of 1,799 Enterococcus faecalis isolates were isolated from inpatients of Gunma University Hospital, Gunma, Japan, between 1992 and 1996. Four hundred thirty-two (22.3%) of the 1,799 isolates had high-level gentamicin resistance. Eighty-one of the 432 isolates were classified and were placed into four groups (group A through group D) with respect to the EcoRI restriction endonuclease profiles of the plasmid DNAs isolated from these strains. The 81 isolates were isolated from 36 patients. For 35 of the 36 patients, the same gentamicin-resistant isolates were isolated from the same or different specimens isolated from the same patient at different times during the hospitalization. For one other patient, two different groups of the isolates were isolated from the same specimen. Groups A, B, C, and D were isolated from 5, 14, 12, and 6 patients, respectively. The strains had multiple-drug resistance. The restriction endonuclease digestion patterns of the E. faecalis chromosomal DNAs isolated from isolates in the same group were also identical. The patients who had been infected with the gentamicin-resistant isolates from each group were geographically clustered on a ward(s). These results suggest that the isolates in each group were derived from a common source and had spread in the ward. The gentamicin-resistant isolates exhibited a clumping response upon exposure to pheromone (E. faecalis FA2-2 culture filtrate). The gentamicin resistance transferred at a high frequency to the recipient E. faecalis isolates by broth mating, and the pheromone-responsive plasmids encoding the gentamicin resistance were identified in these isolates.Enterococcus strains have become a significant cause of nosocomial infections (15, 17, 18, 22, 27). Of the members of the genus Enterococcus, E. faecalis and E. faecium are commonly isolated from humans. These two organisms account for 85 to 95 and 5 to 10% of the strains isolated from clinical infections, respectively. The Enterococcus strains isolated from clinical infections have multiple-drug resistance. The multiple-drug resistance of the enterococci provides these organisms with a selective advantage in the hospital environment. Outbreaks of nosocomial infections caused by enterococcal strains resistant to various drugs have been reported previously (9, 10, 16–18, 23, 28, 29).In a study of clinical isolates from patients in Gunma University Hospital in Gunma, Japan, enterococci were found to be the second most common among the gram-positive bacteria, after Staphylococcus aureus (unpublished data). Of the clinical E. faecalis isolates, most (about 80%) were resistant to tetracycline. Between 30 and 40% of the isolates were resistant to gentamicin or erythromycin. Ampicillin- or vancomycin-resistant strains were not isolated (14, 24). Certain E. faecalis conjugative plasmids confer a mating response to the small sex pheromones secreted by potential recipient cells (1–4, 8, 11). This mating signal induces the synthesis of a surface aggregation substance that facilitates the formation of mating aggregates and plasmid transfer (2–4, 7, 11, 25). Most (60%) of the drug-resistant strains exhibit a clumping response with a culture filtrate of a plasmid-free E. faecalis recipient strain (24), suggesting that the strains harbor a pheromone-responding plasmid.To our knowledge, there is no report concerning nosocomial infection caused by enterococci in Japan. In this report, we describe nosocomial infections in Gunma University Hospital caused by high-level gentamicin-resistant isolates of E. faecalis and isolation of the pheromone-responsive plasmids from the isolates.     

Effect of Prior Experimental Human Enteropathogenic Escherichia coli Infection on Illness following Homologous and Heterologous Rechallenge

Two studies of adult volunteers were performed to determine whether prior enteropathogenic Escherichia coli (EPEC) infection confers protective immunity against rechallenge. In the first study, a naive control group and volunteers who had previously ingested an O55:H6 strain were fed an O127:H6 strain. In the second study, a control group and volunteers who had previously ingested either the O127:H6 strain or an isogenic eae deletion mutant of that strain were challenged with the homologous wild-type strain. There was no significant effect of prior infection on the incidence of diarrhea in either study. However, in the homologous-rechallenge study, disease was significantly milder in the group previously challenged with the wild-type strain. Disease severity was inversely correlated with the level of prechallenge serum immunoglobulin G against the O127 lipopolysaccharide. These studies indicate that prior EPEC infection can reduce disease severity upon homologous challenge. Further studies may require the development of new model systems.

Enteropathogenic Escherichia coli (EPEC) strains are one of several categories of pathogenic E. coli strains that cause diarrhea. EPEC infections are prevalent on six continents (5, 22–24, 28, 43). In many parts of the world, EPEC strains are the most common bacterial cause of diarrhea in infants (7, 21, 43). Disease due to EPEC can be severe, refractory to oral rehydration, protracted, and lethal (3, 14, 21, 45, 48).The pathogenesis of EPEC infection involves three distinct stages, initial adherence, signal transduction, and intimate attachment (12). Initial adherence is associated with the production of a type IV fimbria, the bundle-forming pilus (BFP) (20), that is encoded on the large EPEC adherence factor (EAF) plasmid (50). EPEC uses a type III secretion apparatus to export several proteins, including EspA, EspB, and EspD, that are required for tyrosine kinase-mediated signal transduction within the host cell (17, 25, 30, 31). This signaling leads to phosphorylation and activation of a 90-kDa protein that is a putative receptor for the bacterial outer membrane protein intimin (44). Intimin, the product of the eae gene, is required for intimate attachment of bacteria to the host cell membrane and for full virulence in volunteers (13, 26, 27). The interaction between EPEC and host cells results in the loss of microvilli and the formation of adhesion pedestals containing numerous cytoskeletal proteins (16, 33, 34, 39, 46). This interaction between bacteria and host cells is known as the attaching and effacing effect (40).One of the most striking clinical features of EPEC infections is the remarkable propensity of these strains to cause disease in very young infants. Rare reports of disease in older children and adults usually reflect common-source outbreaks that probably involve large inocula (47, 53). In contrast, in nosocomial outbreaks among neonates, EPEC spreads rapidly by person-to-person contact, apparently involving low inocula (54). The incidence of community-acquired EPEC infection is highest in the first 6 months after birth (4, 7, 21). EPEC infection is also more severe in younger children (8). Infants are more likely to develop diarrhea during the first episode of colonization with EPEC than they are during subsequent encounters (8). Whether the low incidence of EPEC diarrhea in older children and adults is due to acquired immunity or decreased inherent susceptibility is not known.The immune response to EPEC infection remains poorly characterized. It has previously been demonstrated that volunteers convalescing from experimental EPEC infection develop antibodies to the O antigen component of lipopolysaccharide (LPS) of the infecting strain, to intimin, and to type I-like fimbriae (13, 15, 29, 38). Antibodies to common EPEC O antigens are found more often in children of greater than 1 year in age than they are in younger children (42). Breast-feeding is protective against EPEC infection (2, 19, 43, 52). Breast milk contains antibodies against EPEC O antigens and outer membrane proteins and inhibits EPEC adherence to tissue culture cells (6, 9, 49).In an earlier study, it was reported that volunteers infected with EPEC developed antibodies to a 94-kDa outer membrane protein (38). Subsequently, it was determined that this antigen was intimin (26). Interestingly, the lone volunteer in that earlier study who did not have diarrhea after challenge with a wild-type EPEC strain had prechallenge serum antibodies to intimin. This led to the hypothesis that antibodies to intimin are protective against EPEC infection. To test this hypothesis and to test the more general hypothesis that EPEC infection induces protective immunity, two volunteer studies were performed. The first was a heterologous-challenge study performed in 1986, in which volunteers were infected with an O55:H6 EPEC strain and challenged, along with a naive cohort, with an O127:H6 EPEC strain. The second was a homologous-challenge study performed in 1991, in which veterans of a study comparing the virulence of a wild-type EPEC O127:H6 strain with that of an isogenic eae mutant (13) were rechallenged, along with a naive cohort, with the homologous wild-type strain. The availability of new purified antigens allowed us to analyze data from these studies in the context of humoral immune responses.