Acid-induced aggregation propensity of nivolumab is dependent on the Fc

Nivolumab, an anti-programmed death (PD)1 IgG₄ antibody, has shown notable success as a cancer treatment. Here, we report that nivolumab was susceptible to aggregation during manufacturing, particularly in routine purification steps. Our experimental results showed that exposure to low pH caused aggregation of nivolumab, and the Fc was primarily responsible for an acid-induced unfolding phenomenon. To compare the intrinsic propensity of acid-induced aggregation for other IgGs subclasses, tocilizumab (IgG₁), panitumumab (IgG₂) and atezolizumab (aglyco-IgG₁) were also investigated. The accurate pH threshold of acid-induced aggregation for individual IgG Fc subclasses was identified and ranked as: IgG₁?1?2?4. This result was cross-validated by thermostability and conformation analysis. We also assessed the effect of several protein stabilizers on nivolumab, and found mannitol ameliorated the acid-induced aggregation of the molecule. Our results provide valuable insight into downstream manufacturing process development, especially for immune checkpoint modulating molecules with a human IgG₄ backbone.     

Relationships between IgE/IgG4 Epitopes,Structure and Function in Anisakis simplex Ani s 5, a Member of the SXP/RAL-2 Protein Family

Background

Anisakiasis is a re-emerging global disease caused by consumption of raw or lightly cooked fish contaminated with L3 Anisakis larvae. This zoonotic disease is characterized by severe gastrointestinal and/or allergic symptoms which may misdiagnosed as appendicitis, gastric ulcer or other food allergies.The Anisakis allergen Ani s 5 is a protein belonging to the SXP/RAL-2 family; it is detected exclusively in nematodes. Previous studies showed that SXP/RAL-2 proteins are active antigens; however, their structure and function remain unknown.The aim of this study was to elucidate the three-dimensional structure of Ani s 5 and its main IgE and IgG₄ binding regions.

Methodology/Principal Findings

The tertiary structure of recombinant Ani s 5 in solution was solved by nuclear magnetic resonance. Mg²⁺, but not Ca²⁺, binding was determined by band shift using SDS-PAGE. IgE and IgG₄ epitopes were elucidated by microarray immunoassay and SPOTs membranes using sera from nine Anisakis allergic patients.The tertiary structure of Ani s 5 is composed of six alpha helices (H), with a Calmodulin like fold. H3 is a long, central helix that organizes the structure, with H1 and H2 packing at its N-terminus and H4 and H5 packing at its C-terminus. The orientation of H6 is undefined. Regarding epitopes recognized by IgE and IgG4 immunoglobulins, the same eleven peptides derived from Ani s 5 were bound by both IgE and IgG₄. Peptides 14 (L40-K59), 26 (A76-A95) and 35 (I103-D122) were recognized by three out of nine sera.

Conclusions/Significance

This is the first reported 3D structure of an Anisakis allergen. Magnesium ion binding and structural resemblance to Calmodulin, suggest some putative functions for SXP/RAL-2 proteins. Furthermore, the IgE/IgG₄ binding regions of Ani s 5 were identified as segments localized on its surface. These data will contribute towards a better understanding of the interactions that occur between immunoglobulins and allergens and, in turn, facilitate the design of novel diagnostic tests and immunotherapeutic strategies.     

Interaction of ϵ-dinitrophenyl (DNP)-l-lysine with bovine colostral anti-DNP immunoglobulin G1 (IgG1): Concentration independence

Study of the interaction (or interactions) of ?-DNP-l-lysine with bovine colostral anti-DNP IgG₁ using an equilibrium dialysis procedure revealed that the total affinity constant (K_t) decreased with an increase in the antibody concentration. However, the curves in Scatchard and log Q′ plots were superimposable if the data were corrected for nonspecific binding by normal IgG₁. This negative cooperative effect observed with nonspecific IgG₁ was abrogated on enzymatic removal of Fe′. Isolated Fe′ fragments, on the other hand, exhibited the negative cooperative effect on interaction with ?-DNP-l-lysine, thus suggesting that the specific binding and hence the K_t was independent of antibody concentration.     

Indirect plaque-forming cells detected by use of normal mouse serum. I. Normal mouse serum plaque-forming cells are IgA producers.

The identity of indirect plaques developed upon addition of normal mouse serum (NMS) to Jerne test dishes was investigated. NMS plaques were significantly correlated with IgA, IgG_2A and IgG_2B plaques in decreasing order, but not with IgG₁ plaques. With protein A of Staphylococcus aureus as developer of IgG₂ plaques, the nonidentity of NMS plaque-forming cells (PFC) and IgG₂-producing cells could be demonstrated. The subsequent use in different orders of anti-α serum and NMS as developers of indirect plaques suggested that NMS PFC are IgA producers. Blocking experiments with purified myeloma IgA (MOPC 315) supplied further evidence for this idea. Using easily available protein A and NMS as developers of indirect plaques enables collection of more uniform data on the numbers of IgG₂ and IgA PFC than could be obtained with anti-heavy-chain sera.     

Development of Ss-NIE-1 Recombinant Antigen Based Assays for Immunodiagnosis of Strongyloidiasis

Strongyloides stercoralis is a widely distributed parasite that infects 30 to 100 million people worldwide. In the United States strongyloidiasis is recognized as an important infection in immigrants and refugees. Public health and commercial reference laboratories need a simple and reliable method for diagnosis of strongyloidiasis to identify and treat cases and to prevent transmission. The recognized laboratory test of choice for diagnosis of strongyloidiasis is detection of disease specific antibodies, most commonly using a crude parasite extract for detection of IgG antibodies. Recently, a luciferase tagged recombinant protein of S. stercoralis, Ss-NIE-1, has been used in a luciferase immunoprecipitation system (LIPS) to detect IgG and IgG₄ specific antibodies. To promote wider adoption of immunoassays for strongyloidiasis, we used the Ss-NIE-1 recombinant antigen without the luciferase tag and developed ELISA and fluorescent bead (Luminex) assays to detect S. stercoralis specific IgG₄. We evaluated the assays using well-characterized sera from persons with or without presumed strongyloidiasis. The sensitivity and specificity of Ss-NIE-1 IgG₄ ELISA were 95% and 93%, respectively. For the IgG₄ Luminex assay, the sensitivity and specificity were 93% and 95%, respectively. Specific IgG4 antibody decreased after treatment in a manner that was similar to the decrease of specific IgG measured in the crude IgG ELISA. The sensitivities of the Ss-NIE-1 IgG₄ ELISA and Luminex assays were comparable to the crude IgG ELISA but with improved specificities. However, the Ss-NIE-1 based assays are not dependent on native parasite materials and can be performed using widely available laboratory equipment. In conclusion, these newly developed Ss-NIE-1 based immunoassays can be readily adopted by public health and commercial reference laboratories for routine screening and clinical diagnosis of S. stercoralis infection in refugees and immigrants in the United States.     

Compensatory reaction during degeneration of arcuate nucleus dopaminergic neurons in rats

The study has been carried out to verify the authors’ hypothesis that degeneration of dopaminergic (DA-ergic) neurons of the hypothalamic tuberoinfundibular system and concomitant development of hyperprolactinemia are accompanied by involvement of compensatory synthesis of dopamine (DA) by non-dopaminergic neurons expressing single complementary enzymes of synthesis of this neurotransmitter. Degeneration of DA-ergic neurons was produced by a stereotaxic injection into the brain lateral ventricles of 6-hydroxydopamine (6-HDA)—a specific neurotoxin of DA-ergic neurons. 14 and 45 days after the toxin administration there were determined concentration of prolactine in peripheral blood by methods of immunoenzyme and radioimmunological analyses as well as the DA amount in the arcuate nucleus by the method of highly efficient liquid chromatography with electrochemical detection. In a part of the animals, sections were prepared from the mediobasal hypothalamus (arcuate nucleus and medial eminence) and perfused with Krebs—Ringer medium; then the DA concentration was determined in the sections and in the incubation medium. 14 days after the neurotoxin administration there were revealed an increase of blood prolactine concentration and a decrease of DA concentration in the arcuate nucleus in vivo as well a decrease of the total DA amount in the sections and incubation medium in experiments in vitro. 45 days after the neurotoxin administration, all the above parameters returned to the normal level. Thus, the obtained data indicate that the hyperlactinemia and DA deficit appearing during degeneration of the arcuate nucleus DA-ergic neurons seem to be compensated due to an enhancement of DA synthesis by non-dopaminergic monoenzyme neurons of arcuate nucleus.     

Tumor localization of Lewis lung carcinoma with radiolabeled monoclonal antibodies

Summary Mouse 6D6 IgG_2a and 5B5 IgM monoclonal antibodies which specifically bind murine lung carcinoma cells (3LL cells) were injected to healthy and tumor-bearing mice. In vivo localization was analyzed by counting the tissue radioactivity and by external gamma ray scintigraphy at various times after IV injection of ¹²⁵I- or ¹³¹I-labeled antibodies. The clearance of the two monoclonal antibodies was not modified by the presence of the tumor, and the 6D6 IgG_2a was cleared at a rate slower than the 5B5 IgM. Both antibodies gave a high specific uptake at the tumor level; the tumor-to-healthy tissue ratios were higher with the 6D6 IgG_2a than the 5B5 IgM; unspecific mouse immunoglobulins (IgG₂) did not localize in the tumor. The amount of 6D6 IgG_2a antibody still associated with the tumor after 2 days following IV injection was 10 times higher than that of 5B5 IgM, and was still high enough to localize the tumor after 5 days.Imaging experiments confirmed the ability of 6D6 IgG_2a to detect the presence of tumor cells. The targeting kinetics determined by computer analysis of camera images indicated a rapid targeting of antibodies in tumor with a maximal concentration after 4–6 h; after 48 h the background was quite low and the 6D6 IgG_2a appeared to be specifically localized in the tumor.     

Adjuvant and immunogenic properties of bacterial lipopolysaccharide in IgE and IgG1 antibody formation in mice

The ability of Gram-negative lipopolysaccharide (LPS) to function as an adjuvant and as an antigen in IgE and IgG₁ immune responses in mice was investigated. LPS failed to induce LPS-specific IgE or IgG₁ under a variety of experimental conditions. Both isolated LPS and whole heat-killed bacteria were capable of enhancing IgE and IgG₁ antibody formation to a protein antigen, egg albumin (EA). The LPS-induced anti-EA, IgE, and IgG₁ antibody titers exhibited a cycling phenomenon with time. In the presence of LPS, IgE, and IgG₁ antibodies specific for EA did not occur in athymic nude (BALB/c-nu/nu) mice, demonstrating the inability of LPS to substitute for the stringent requirement for T cells in homocytotropic antibody formation.     

Modulation of the immune response by passive antibodies. III. Effects of IgG1 and IgG2 anti-carrier antibodies.

The effects of IgG₁ and IgG₂ anti-carrier antibodies were studied on cellular and humoral reactions induced by immunization with a hapten-carrier complex. IgG₁ was shown to depress both delayed hypersensitivity reactions (DHR) to the carrier and anaphylaxis to the hapten whereas IgG₂ had no activity. A mixture of IgG₁ and IgG₂ depressed only DHR to the carrier. The modulating effects of passive anti-carrier antibodies were shown to depend on their immunoglobulin class and the concentration used.     

Cellular subpopulations in the expression of IgG1.

Consistent with the surface display of IgG₁, antigen-primed B cells are sensitive to functional elimination by anti-Ig and C. This depletion of IgG₁ responses, assayed in adoptive transfer, is accompanied by increased responses of other isotypes, a phenomenon termed compensation. An analysis of the tertiary response reveals that cells sensitive to functional elimination are precursors to memory cells as well as plaque-forming cells. To investigate control mechanisms governing preferential expression of IgG₁, and the compensation active after elimination of IgG₁, increased numbers of carrier-primed cells were transferred and antigen dose was increased to adoptive recipients. Doses of carrier-primed cells or antigen were found which allowed maximal expression of both IgG₁ and non-IgG₁ isotypes. In recipients of anti-Ig and C-treated B cells, increased numbers of carrier-primed cells did not further increase the compensatory expression of non-IgG₁ isotypes. A discussion is presented of compensation as a result of limiting inductive signals to B cells.     

Antagonistic pleiotropic effects of nitric oxide in the pathophysiology of Parkinson's disease

Abstract

Sporadic Parkinson's disease (PD) is a geriatric disorder with unknown etiology, specifically affecting the nigrostriatal dopaminergic (DA-ergic) pathway of the brain. Amongst several contributing factors, nitric oxide (NO?) is considered to inflict injury to DA-ergic neurons, and to influence PD progression. Supportive evidence for this comes from animal models of PD, where inhibitors of NO? synthase (NOS) are found to protect against DA-ergic neuronal death, and NOS-deficient mice are found to be resistant to PD-producing neurotoxins. Presence of nitrated proteins and upregulated levels of NOS in human postmortem PD brain samples have rendered further support to this contention. While NO? from neuronal NOS contributes to neurodegeneration in PD, NO? produced by inducible NOS from proliferating microglia as inflammatory responses to neuronal insults are suggested to mediate the disease progression. Another view that NO? in small doses serves as a neuroprotective agent in the brain is also discussed, in light of experimental evidence available in vitro and in vivo. This view is based on the argument that NO? could form harmless nitrites and nitrates on reaction with endogenously produced reactive oxygen species (ROS) within the cells. This review essentially discusses the possibilities of considering NO? as a secondary response of DA-ergic cell death, while oxidative stress is the primary cause. Once neurons undergo death processes following uncontrolled oxidative insult, the resulting gliosis-mediated NO? accelerates the events as a secondary mediator. Since the time of initiation of DA-ergic cell death cannot be predicted, NO? could be an ideal molecular target to halt the disease progression.     

Higher cytolytic efficiency of an IgG2 alpha than of an IgG1 monoclonal antibody reacting with the same (or spatially close) determinant on a human high-molecular-weight melanoma-associated antigen

Serological and immunochemical assays showed that the monoclonal antibody (MoAb) 225.28S, an IgG_2α, and the MoAb 653.40S, an IgG₁, react with the same (or spatially close) antigenic determinant expressed on a set of molecules carrying a high-molecular-weight human melanoma-associated antigen. Neither monoclonal antibody mediates complement-dependent lysis of cultured melanoma cells, but both of them specifically mediate lysis of target cells in an antiglobulin cytotoxic assay and in an antibody-dependent cell-mediated cytotoxicity (ADCC) assay. In the latter two assays the IgG_2α displays a higher lytic activity than the IgG₁. The differential lytic activity of the IgG_2α and IgG₁ monoclonal antibodies was detected also when the sensitivity of the ADCC assay was increased either by boosting the cytolytic activity of the effector cells or by enhancing the susceptibility to lysis of target cells.     

Age-related prevalence of antibodies to infective larvae ofWuchereria bancrofti in normal individuals from a filaria-endemic region

Antibody isotypic levels (IgM, IgE and IgG subclasses) to infective larvae (L₃) ofWuchereria bancrofti were measured in 104 normal individuals from a filaria-endemic region in Orissa. The titres of antibodies were considerably higher in adults (n = 25, 25.1± 3.8 year) than in children (n = 52, 7.1 ± 2.1 year). Young children (n = 14) less than four years were seronegative to all isotypes other than IgM, the sero-conversion to which was achieved in the children (n=15) by the age of 7.5±1.2 years. The prevalence of other isotypes increased with age and reached a maximum in early adulthood (18.6 ±1.6 years), which persisted in older adults (> 30 years). However, the increase in IgG₃ prevalence with age was less marked. IgG₂ was detected only after 10 years of age. Compared to the high prevalence (100%) of IgM, IgE, IgG₁, and IgG₂, in adults. IgG₃ and IgG₄ prevalences were low, 35% and 28% respectively. IgA level to L₃ antigen was found to be extremely low even in adults. These data indicate that the prevalence of L₃ antibodies was different for different isotypes and the acquisition of antibody response essentially followed an age dependent pattern.     

Mesangial Cell-Binding Activity of Serum Immunoglobulin G in Patients with Lupus Nephritis

In vitro data showed that immunoglobulin G (IgG) from patients with lupus nephritis (LN) could bind to cultured human mesangial cells (HMC). The clinical relevance of such binding was unknown. Binding of IgG and subclasses was measured in 189 serial serum samples from 23 patients with Class III/IV±V LN (48 during renal flares, 141 during low level disease activity (LLDA)). 64 patients with non-lupus glomerular diseases (NLGD) and 23 healthy individuals were used as controls. HMC-binding was measured with cellular ELISA and expressed as OD index. HMC-binding index of total IgG was 0.12±0.09, 0.36±0.25, 0.59±0.37 and 0.74±0.42 in healthy subjects, NLGD, LN patients during LLDA, and LN flares respectively (P = 0.046, LN flare vs. LLDA; P<0.001, for healthy controls or NLGD vs. LN during flare or LLDA). Binding of serum IgG₁ to HMC was 0.05±0.05, 0.15±0.11, 0.41±0.38 and 0.55±0.40 for the corresponding groups respectively (P = 0.007, LN flare vs. remission; P<0.001, for healthy controls or NLGD vs. LN during flare or remission). IgG₂, IgG₃ and IgG₄ from patients and controls did not show significant binding to HMC. Total IgG and IgG₁ HMC-binding index correlated with anti-dsDNA level (r = 0.26 and 0.39 respectively, P<0.001 for both), and inversely with C3 (r = −0.17 and −0.45, P<0.05 for both). Sensitivity/specificity of total IgG or IgG₁ binding to HMC in predicting renal flares were 81.3%/39.7% (ROC AUC 0.61, P = 0.03) and 83.8%/41.8% (AUC 0.63, P = 0.009) respectively. HMC-binding by IgG_1, but not total IgG, correlated with mesangial immune deposition in LN renal biopsies under electron microscopy. Our results showed that binding of serum total IgG and IgG₁ in LN patients correlates with disease activity. The correlation between IgG₁ HMC-binding and mesangial immune deposition suggests a potential pathogenic significance.     

Strain differences in tolerance induction to human gamma-globulin subclasses: dependence on macrophages.

BALB/c and DBA/2 mice differ with respect to ease of tolerance induction with HGG, BALB/c mice being the resistant strain. When tested for susceptibility to tolerance induction with individual IgG subclasses, both strains were easily rendered unresponsive with IgG₁ and IgG₂ and less so with IgG₄. A strain difference appeared with IgG₃, where only BALB/c mice showed complete resistance to tolerance induction. Mixtures of the IgG subclasses Showed that IgG₁ and IgG₂ accounted for most of the tolerance to whole HGG seen in both strains, while addition of IgG₃ to the mixture made DBA/2 completely tolerant but reversed the trend toward tolerance in the BALB/c mice. By rosette assay it was found that BALB/c macrophages had receptors for IgG₃ (and to a lesser extent for IgG₄). These results are consistent with the hypothesis that resistance to tolerance induction with HGG is dependent on the presence of a receptor on the macrophage for a minor IgG subclass.     

Effects of excess thyroid hormone on cell death,cell proliferation,and new neuron incorporation in the adult zebra finch telencephalon

Widespread telencephalic neuronal replacement occurs throughout life in birds. We explored the potential relationship between thyroxine (T₄) and cell turnover in the adult male zebra finch. We found that many cells in the zebra finch brain, including long‐projection neurons in the high vocal center (HVC), stained positively with an antibody to thyroid hormone receptors (TR). Labeling was generally weak in the ventricular zone (VZ) that gives rise to new neurons but some proliferative VZ cells and/or their progeny, identified by [³H]‐thymidine labeling, co‐labeled with anti‐TR antibody. Acute T₄ treatment dramatically increased the number of pyknotic and TUNEL‐positive cells in HVC and other telencephalic regions. In contrast, degenerating cells were never observed in the archistriatum or sub‐telencephalic regions, suggesting that excess T₄ augments cell death selectively in regions that show naturally occurring neuronal turnover. VZ mitotic activity was not altered shortly after acute T₄ treatment at a dosage that stimulated cell death, although [³H]‐labeling intensity per cell was slightly reduced. Moreover, the incorporation rates for neurons formed shortly before or after acute hormone treatment were no different from control values. Chronic T₄ treatment resulted in a reduction in the total number of HVC neurons. Thus, hyperthyroidism augmented neuronal death, which was not compensated for by neuronal replacement. Collectively, these results indicate that excess T₄ affects adult neuronal turnover in birds, and raises the possibility that thyroxine plays an important role in the postnatal development of the avian brain and vocal behavior. © 2002 Wiley Periodicals, Inc. J Neurobiol 51: 323–341, 2002     

Detection of disease specific sialoglycoconjugate specific antibodies in bronchoalveolar lavage fluid of non-small cell lung cancer patients

In this study, we report the presence of significantly higher level of GM3 specific IgG antibodies (IgG_TL) in the bronchoalveolar lavage fluid obtained from tumor bearing lung of non-small cell lung cancer (NSCLC) patients as compared to other non-neoplastic controls. The antibodies were isolated using DEAE-cellulose anion exchange chromatography and molecular weight of the subunits of IgG_TL was confirmed in SDS-PAGE. IgG_TL revealed high specificity to GM3 and the IgG distribution was confined to IgG1. Furthermore, IgG_TL showed strong reactivity with NSCLC cell lines as well as the tissue biopsies and cells obtained from fine needle aspirations of NSCLC patients. A 66 kDa membrane glycoprotein of NSCLC cell lines was found to interact specifically with IgG_TL, the intensity of which was drastically reduced in presence of GM3. Further, binding of Maackia amurensis agglutinin [specific for NeuAcα(2→3)Gal unit , the same disaccharide unit also known to be present in GM3] to the 66 kDa band confirmed it to be a sialoglycoprotein in nature. IgG_TL could not show any reactivity to alkaline borohydrate treated or periodate oxidised membrane fractions, suggesting the probable involvement of the carbohydrate moiety of the 66 kDa glycoprotein in the interaction with IgG_TL. Thus, the 66 kDa sialoglycoprotein seems to be the NSCLC specific sialoglycoconjugate. Taken together, IgG_TL antibodies may have the potential to serve as a unique probe for detail investigation of NSCLC specific cell surface sialoglycoconjugate. Further, due to high specificity of IgG_TL to GM3, it may be possible to develop a simple alternative diagnostic approach (GM3-ELISA) for NSCLC.     

Modulation of mouse anti-SRBC antibody response by placental extracts

Mouse placental extracts (PE) and corresponding Sephadex G-200 fractions were administered to isogeneic CBA mice along with an optimal immunizing dose of SRBC. Spleen cells were harvested 8 days later and transferred to CBA recipients, subsequently immunized with SRBC. The immunoregulatory activity of spleen cells from PE-treated donors was compared to cells from liver extract (LE)-treated controls or from mice immunized with SRBC only, using Cunningham's PFC direct and indirect tests. Within the dose range used, selective modulatory activities were obtained with cells from PE, but not from LE, treated mice, the latter being comparable to cell transfer effects from donors immunized with SRBC only. Spleen cells from animals injected with low doses of PE (0.25 to 4 mg per mouse) added to immunizing SRBC had a suppressive effect on the primary IgM response of recipients immunized against SRBC. In contrast, when SRBC were given to donor animals with higher doses of PE (8 to 13 mg), transferred spleen cells potentiated the IgM response of the recipients. These opposite suppressive and potentiating activities were found in distinct Sephadex G-200 fractions of 40 and 60 kDa, respectively. When the effect of PE treatment was tested within the same animal, the indirect secondary PFC response following a challenge with SRBC was significantly modified. We observed an overall suppression of the different isotypes after treatment with lower doses of PE or with its 40-kDa fraction. PE doses of 0.5 to 2 mg resulted in a stronger inhibition of IgM than IgG₁ production. This phenomenon was also obtained with the 40 KDa fraction. IgG₂ responses were significantly reduced by all doses of this fraction. In contrast, all doses of the 60-kDa fraction gave a strong stimulation of IgG₂ and IgM responses and a constant suppression of the IgG₁ response. This shows a clear dissociation between IgG₁ and C'-fixing (IgM, IgG₂) antibody classes as far as the influence of placental substances is concerned in their regulation. These data emphasize the relevance of isogeneic placental products as a useful physiological material capable of modulating xenogeneic immune responses (as well as allogeneic systems).     

Heligmosomoides polygrus (equals nematospiroides dubius): humoral and intestinal immunologic responses to infection in mice

Measurements of serum IgG₁, IgG_2a, IgM, and IgA levels and antibody titers in these immunoglobulin classes were made at intervals after initial infection and challenge infection of mice immunized by two or three previous infections. Identical measurements were made on the content of the small intestine in mice which had been exposed to the same infection schedule. Sections of small intestine taken after initial infection and challenge infection were examined by the fluorescent antibody technique for changes in populations of immunoglobulin-containing cells and by routine histologic procedures for histopathologic changes.In serum, only IgG₁ was consistently increased after initial infection, and antibody in IgG₁ was detected within the first 2 wk of infection. In immunized animals, only IgG₁ and antibody of this class always responded to challenge infection, although antibody in other immunoglobulin classes was detected.IgA concentration of the intestinal content did not differ significantly after initial infection or challenge infection of immunized mice. Immunized mice had about twice the IgG₁ concentration in intestinal content as singly infected animals. No intestinal antibody was detected after initial infection; only IgG₁ antibody was detected in the intestinal content of immunized and challenged mice.Cell infiltrates in the intestinal mucosa and submucosa of immunized animals contained numerous IgG₁-containing cells. Mast cells and globular leukocytes were observed in the intestine of immunized animals.