Longitudinal study of serum neutralization of HIV-1 in infected plasma donors.

Earlier studies provided suggestive evidence about the effectiveness of passive immunotherapy for AIDS patients using plasma donated by healthy HIV-1 infected individuals and revealed beneficial effects of plasmapheresis for the immune system of the donors. In this study, anti-HIV-1 neutralizing activity in 31 healthy HIV-1 infected donors of plasma participating in a passive immunotherapy study was investigated as a function of time. Average studied period was 33 months. Using the highly cytopathic HIV-1 NDK strain and MOLT4 cells, it was shown by means of syncytia formation inhibition assay and polyclonal HIV-1 antigen-capture assay that viral neutralizing titers tend either to remain unchanged or increase over time. These findings support the notion that the immune system is not affected adversely in HIV-1 infected plasma donors and lend further support to the feasibility of passive immunotherapy for AIDS patients.

A human myeloma cell line suitable for the generation of human monoclonal antibodies.

Ever since monoclonal antibodies were produced in 1975 with mouse myeloma cells there has been interest in developing human myeloma cultures for the production of monoclonal antibodies. However, despite multiple attempts, no human myeloma line suitable for hybridoma production has been described. Here we report the derivation of a hypoxanthine-aminopterin-thymidine-sensitive and ouabain-resistant human myeloma cell line (Karpas 707H) that contains unique genetic markers. We show that this line is useful for the generation of stable human hybridomas. It can easily be fused with ouabain-sensitive Epstein-Barr virus-transformed cells as well as with fresh tonsil and blood lymphocytes, giving rise to stable hybrids that continuously secrete very large quantities of human immunoglobulins. The derived hybrids do not lose immunoglobulin secretion over many months of continuous growth. The availability of this cell line should enable the in vitro immortalization of human antibody-producing B cells that are formed in vivo. The monoclonal antibodies produced may have advantages in immunotherapy.

Molecular characterization of human monoclonal antibodies derived from fusions of tonsil lymphocytes with a human myeloma cell line.

Recently a new human myeloma cell line (Karpas 707H) has been developed for the efficient generation of stable human hybridomas. Here we describe the first molecular characterization of human monoclonal antibodies (MAbs) produced by a human counterpart to mouse myeloma cells. We studied 30 of the hybridomas generated by fusions to tonsil lymphocytes by DNA sequencing of rearranged V-genes, and have analyzed germ-line diversity, somatic hypermutation, and heavy- and light-chain pairings. Our results suggest that the hybridoma-derived antibodies are representative of antibodies from populations of human lymphocytes and at different stages in the maturation of the response; the use of Karpas 707H myeloma for human hybridoma fusions may therefore provide a valuable tool for analysis of the human antibody responses.

Phase 1 and phase 2 studies of Salmonella enterica serovar paratyphi A O-specific polysaccharide-tetanus toxoid conjugates in adults, teenagers, and 2- to 4-year-old children in Vietnam.

Salmonella enterica serovar Paratyphi A O-specific polysaccharide (O-SP) was activated with 1-cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP) and bound to tetanus toxoid (TT) with adipic acid dihydrazide as a linker (SPA-TT(1)) or directly (SPA-TT(2)). In mice, these two conjugates elicited high levels of immunoglobulin G (IgG) anti-lipopolysaccharide (LPS) in serum with bactericidal activity (E. Konadu, J. Shiloach, D. A. Bryla, J. B. Robbins, and S. C. Szu, Infect. Immun. 64:2709-2715, 1996). The safety and immunogenicity of the two conjugates were then evaluated sequentially in Vietnamese adults, teenagers, and 2- to 4-year-old children. None of the vaccinees experienced significant side effects, and all had preexisting LPS antibodies. At 4 weeks after injection, there were significant increases of the geometric mean IgG and IgM anti-LPS levels in the adults and teenagers: both conjugates elicited a greater than fourfold rise in the IgG anti-LPS level in serum in >/=80% of the volunteers. SPA-TT(2) elicited slightly higher, though not statistically significantly, levels of IgG anti-LPS than did SPA-TT(1) in these age groups. Accordingly, only SPA-TT(2) was evaluated in the 2- to 4-year-old children. On a random basis, one or two injections were administered 6 weeks apart to the children. No significant side effects were observed, and the levels of preexisting anti-LPS in serum were similar in children of all ages. A significant rise in the IgG anti-LPS titer was elicited by the first injection (P = 0.0001); a second injection did not elicit a booster response. Representative sera from all groups had bactericidal activity that could be adsorbed by S. enterica serovar Paratyphi A LPS.

Safety and immunogenicity of Vi conjugate vaccines for typhoid fever in adults, teenagers, and 2- to 4-year-old children in Vietnam.

The capsular polysaccharide of Salmonella typhi, Vi, is an essential virulence factor and a protective vaccine for people older than 5 years. The safety and immunogenicity of two investigational Vi conjugate vaccines were evaluated in adults, 5- to 14-year-old children, and 2- to 4-year-old children in Vietnam. The conjugates were prepared with Pseudomonas aeruginosa recombinant exoprotein A (rEPA) as the carrier, using either N-succinimidyl-3-(2-pyridyldithio)-propionate (SPDP; Vi-rEPA(1)) or adipic acid dihydrazide (ADH; Vi-rEPA(2)) as linkers. None of the recipients experienced a temperature of >38.5 degrees C or significant local reactions. One injection of Vi-rEPA(2) into adults elicited a geometric mean (GM) increase in anti-Vi immunoglobulin G (IgG) from 9.62 enzyme-linked immunosorbent assay units/ml (EU) to 465 EU at 6 weeks; this level fell to 119 EU after 26 weeks. In the 5- to 14-year-old children, anti-Vi IgG levels at 6 weeks elicited by Vi-rEPA(2), Vi-rEPA(1), and Vi were 169, 22.8, and 18.9 EU, respectively (P = 0.0001 for Vi-rEPA(1) and Vi with respect to Vi-rEPA(2)). At 26 weeks, the anti-Vi IgG levels for recipients of Vi-rEPA(2), Vi-rEPA(1), and Vi were 30.0, 10.8, and 13.4 EU, respectively (P < 0.001 for Vi-rEPA(1) and Vi with respect to Vi-rEPA(2)); all were higher than the preinjection levels (P = 0. 0001). Vi-rEPA(2) also elicited the highest anti-Vi IgM and IgA levels of the three vaccines. In the 2- to 4-year-old children at 6 weeks following the first injection, Vi-rEPA(2) elicited an anti-Vi IgG level of 69.9 EU compared to 28.9 EU for Vi-rEPA(1) (P = 0.0001). Reinjection increased Vi antibody levels from 69.9 to 95.4 EU for Vi-rEPA(2) and from 28.9 to 83.0 EU for Vi-rEPA(1). At 26 weeks, anti-Vi IgG levels remained higher than those at preinjection (30.6 versus 0.18 for Vi-rEPA(2) and 12.8 versus 0.33 for Vi-rEPA(1); P = 0.0001 for both). Vi vaccine is recommended for individuals of 5 years of age or older. In the present study, the GM level of anti-Vi IgG elicited by two injections of Vi-rEPA(2) in the 2- to 4-year-old children was higher than that elicited by Vi in the 5- to 14-year-old children (30.6 versus 13.4; P = 0.0001). The safety and immunogenicity of the Vi-rEPA(2) conjugate warrant further investigation.

Safety and immunogenicity of Shigella sonnei and Shigella flexneri 2a O-specific polysaccharide conjugates in children.

O-specific polysaccharide conjugates of shigellae were safe and immunogenic in young adults, and a Shigella sonnei conjugate conferred protection [1-3]. Shigellosis is primarily a disease of children; therefore, the safety and immunogenicity of S. sonnei and Shigella flexneri 2a conjugates were studied in 4- to 7-year-old children. Local and systemic reactions were minimal. The first injection of both conjugates elicited significant rises in geometric mean levels of serum IgG only to the homologous lipopolysaccharide (LPS) (S. sonnei, 0.32-8.25 ELISA units [EU]; S. flexneri 2a, 1.15-20.5 EU; P<.0001). Revaccination at 6 weeks induced a booster response to S. flexneri 2a LPS (20.5-30.5 EU, P=.003). Six months later, the geometric mean levels of IgG anti-LPS for both groups were higher than the prevaccination levels (P<.0001). Similar, but lesser, rises were observed for IgM and IgA anti-LPS. The investigational Shigella conjugates were safe and immunogenic in children and merit evaluation of their efficacy.

Differential expression of Vibrio vulnificus capsular polysaccharide.

Vibrio vulnificus is a human pathogen whose virulence has been associated with the expression of capsular polysaccharide (CPS). Multiple CPS types have been described; however, virulence does not appear to correlate with a particular CPS composition. Reversible-phase variation for opaque and translucent colony morphologies is characterized by changes in CPS expression, as suggested by electron microscopy of cells stained nonspecifically with ruthenium red. Isolates with opaque colony morphologies are virulent and appear to be more thickly encapsulated than naturally occurring translucent-phase variants, which have reduced, patchy, or absent CPS. Previously, we have shown that the virulence of translucent-phase variants was intermediate between opaque-phase variants and acapsular transposon mutants, suggesting a correlation between virulence and the amount of CPS expressed. In the present study, CPS expression of phase variants and genetically defined mutants of V. vulnificus M06-24/O was examined by using a CPS-specific monoclonal antibody with an enzyme-linked immunosorbent assay, flow cytometry, and immunoelectron microscopy. Semiquantitative analyses of CPS expression correlated well among these assays, confirming that the translucent-phase variant was intermediate in CPS expression and retained type I CPS-specific epitopes. Cell surface expression of CPS varied with the growth phase, increasing during logarithmic growth and declining in stationary culture. Significantly greater CPS expression (P = 0.026) was observed for cells grown at 30 degrees C than for those at 37 degrees C. These studies confirm that phase variation and virulence in V. vulnificus correlate with the amount of CPS expressed and demonstrate the fluidity of bacterial polysaccharide expression in response to environmental conditions.

Identification of common lipooligosaccharide types in isolates from patients with otitis media by monoclonal antibodies against nontypeable Haemophilus influenzae 9274.

Twenty-one murine monoclonal antibodies (MAbs) were induced by nontypeable Haemophilus influenzae (NTHi) 9274. Nineteen MAbs were specific for the lipooligosaccharide (LOS) as determined by enzyme-linked immunosorbent assay (ELISA) and Western blot analysis. When the MAbs were assayed with five LOS prototype strains by ELISA, all bound to strain 3198 LOS (type III), while six of the MAbs were also reactive with LOSs from strain 1479 (type I), 5657 (type IV), or 7502 (type V). Ten MAbs had complement-mediated bactericidal activity, and three MAbs were opsonophagocytic against the homologous strain. Five LOS MAbs with different specificities were used to analyze 155 NTHi clinical isolates from the United States and from Japan. These isolates were classified into nine groups by ELISA. Only four isolates (2.6%) were not recognized by any of the five MAbs. Most of the isolates (91.6%) were in four groups which bound three of the five MAbs. One of three MAbs, 6347C11, had strong activity against the homologous strain and was also bactericidal to 45 clinical isolates (29%) which belonged to the four common patterns (25 belonged to pattern 1). These data indicate that these MAbs can be used for LOS typing in which almost all NTHi strains can be typed according to the LOS antigenicity. Among NTHi, at least one conserved LOS epitope which is a target of bactericidal antibodies exists. We conclude that strain 9274 LOS, which is the target for bactericidal antibodies, is a candidate for LOS-based NTHi vaccines.

Differential posttranslational processing confers intraspecies variation of a major surface lipoprotein and a macrophage-activating lipopeptide of Mycoplasma fermentans.

The malp gene of Mycoplasma fermentans is shown to occur in single copy but to encode two discrete translated forms of lipid-modified surface protein that can be differentially expressed on isolates within this species: MALP-2, a 14-amino-acid (2-kDa) lipopeptide with potent macrophage-stimulatory activity (P. F. Mühlradt, M. Kiess, H. Meyer, R. Süssmuth, and G. Jung, J. Exp. Med. 185:1951-1958, 1997), and MALP-404, an abundant, full-length (404-amino-acid) surface lipoprotein of 41 kDa, previously designated P41 (K. S. Wise, M. F. Kim, P. M. Theiss, and S.-C. Lo, Infect. Immun. 61:3327-3333, 1993). The sequences, transcripts, and translation products of malp were compared between clonal isolates of strains PG18 (known to express P41) and II-29/1 (known to express high levels of MALP-2). Despite conserved malp DNA sequences containing full-length open reading frames and expression of full-length monocistronic transcripts in both isolates, Western blotting using a monoclonal antibody (MAb) to the N-terminal MALP-2 peptide revealed marked differences in the protein products expressed. Whereas PG18 expressed abundant MALP-404 with detectable MALP-2, II-29/1 revealed no MALP-404 even in samples containing a large comparative excess of MALP-2. Colony immunoblots with the MAb showed uniform surface expression of MALP-2 in II-29/1 populations. A second MAb to an epitope of MALP-404 outside the MALP-2 sequence predictably failed to stain II-29/1 colonies but uniformly stained PG18 populations. Collectively, these results provide evidence for novel posttranscriptional (probably posttranslational) processing pathways leading to differential intraspecies expression of a major lipoprotein, and a potent macrophage-activating lipopeptide, on the surface of M. fermentans. In the course of this study, a striking conserved motif (consensus, TD-G--DDKSFNQSAWE--), designated SLA, was identified in MALP-404; this motif is also distributed among selected lipoproteins and species from diverse bacterial genera, including Bacillus, Borrelia, Listeria, Mycoplasma, and Treponema. In addition, malp was shown to flank a chromosomal polymorphism. In eight isolates of M. fermentans examined, malp occurred upstream of an operon encoding the phase-variable P78 ABC transporter; but, in three of these isolates, a newly discovered insertion sequence, IS1630 (of the IS30 class), was located between these genes.

Delineation of the breakpoint at 18q21.1 in a cell line (Karpas1106) derived from mediastinal B-cell lymphoma by fluorescence in situ hybridization with multiple YAC clones.

The breakpoint of the 18q21 translocation of B-cell-non-Hodgkin's lymphoma (NHL) cell line Karpas1106P was delineated by fluorescence in situ hybridization (FISH). Karpas1106P was derived from mediastinal lymphoblastic B-cell lymphoma and exhibited the immunophenotype characteristic of marginal-zone B-cell lymphoma (MZL): smIg+, pan-B antigen+, CD5-, CD10- and CD23-. The original G-banded karyotype showed a complex translocation containing t(X;18;13)(q28;q21;q12.1). Double-color FISH (DCFISH) with whole-chromosome-painting (WCP) probes for chromosomes X, 13 and 18, and 18q-specific yeast artificial chromosome (YAC) clones defined t(X;18;13) as ider(X)t(X;18; 13)(q28;q 12.3q21.1;q12.1). The immunoglobulin-heavy-chain (IgH) gene was not involved in the chromosomal translocation as detected by DCFISH with VH and Cgamma probes. By using contiguous YAC clones mapped from 18q12.3 to q21.1, we identified a YAC clone y852H2 with its breakpoint at 18q21.1. In Karpas1106P, the distal part of chromosome 18 from the breakpoint (18q21.1-qter) was deleted, showing loss of heterozygosity of this region. In addition, the chromosomal segment 18q21.1 was duplicated and inserted to ider(X)t(X;18; 13) between Xq28 and 13q12.1 with maintaining its original orientation. The DNA sequence of the breakpoint region contained in y852H2 can serve as a candidate locus for further molecular dissection to identify the causative gene of MZL.

How to prolong the effects of combination therapy for HIV.

Infection with HIV leads to AIDS and death in about 90% of patients within ten years. The first generation of anti-HIV drugs inhibited the viral enzyme reverse transcriptase (RT); but long-term studies have revealed side-effects and a high rate of emergence of drug-resistant HIV mutants. The more recent combination of two anti-RT drugs and a protease inhibitor appears to be more promising: approximately 75% of AIDS patients benefit. However, increasing numbers of treatment failures from toxicity and drug-resistant mutants are emerging. Passive immunotherapy (PIT) is a non-toxic form of treatment based on the neutralization of HIV with antibody-rich plasma from healthy HIV-positive individuals. Studies show it can benefit AIDS patients. Here, we suggest that, in combination with anti-HIV drugs, PIT could reduce some of the toxicity of the latter and limit the emergence of drug-resistant HIV strains. In addition, regular plasma donation seems to be beneficial to the donors.

Passive immunotherapy in advanced HIV infection and therapeutic plasmapheresis in asymptomatic HIV-positive individuals: a four-year clinical experience.

We have been treating patients with advanced HIV disease using passive immunotherapy (PIT). Earlier studies of PIT which have been published concerned relatively short periods of treatment: our study is by far the longest and reports also on the long-term effects of plasmapheresis on healthy HIV-infected individuals. Fifty-nine patients with an average CD4+ T-cell count of 55 per cu.mm. at baseline were transfused at monthly intervals with 500 ml of hyperimmune plasma. No disease progression or death occurred among the 8 asymptomatic patients under the treatment, which lasted for 36.25 months on average. Seven of the 15 ARC patients progressed to AIDS but none died in an average period of 25.9 months. Seven of the 36 symptomatic AIDS patients with advanced disease died in an average period of 19.6 months. PIT appears to be nontoxic and to have beneficial effects lasting at least four years under continuous treatment. It probably delays disease progression in ARC and AIDS patients, and almost certainly does so in asymptomatic late HIV infection with a very low CD4+ T-cell count. None of the 51 donors suffered adverse effects, nor did any progress to ARC or AIDS in an average period of 30.1 months. Their laboratory parameters indicated a nearly stable condition: in particular, their average CD4+ T-cell count rose from 478 to 498. The study of our plasma donors indicated that repeated and frequent plasma donation by asymptomatic HIV-infected individuals could delay disease progression, although further studies are needed to investigate this.

On the antigenic determinants of the lipopolysaccharides of Vibrio cholerae O:1, serotypes Ogawa and Inaba.

Monoclonal, murine IgG1s S-20-4, A-20-6, and IgA 2D6, directed against Vibrio cholerae O:1 Ogawa-lipopolysaccharide exhibited the same fine specificities and similar affinities for the synthetic methyl alpha-glycosides of the (oligo)saccharide fragments mimicking the Ogawa O-polysaccharide (O-PS). They did not react with the corresponding synthetic fragments of Inaba O-PS. IgG1s S-20-4 and A-20-6 have absolute affinity constants for synthetic Ogawa mono- to hexasaccharides of from approximately 10(5) to approximately 10(6) M-1. For IgG1s S-20-4, A-20-6, and IgA 2D6, the nonreducing terminal residue of Ogawa O-PS is the dominant determinant, accounting for approximately 90% of the maximal binding energy shown by these antibodies. Binding studies of derivatives of the Ogawa monosaccharide and IgGs S-20-4 and A-20-6 revealed that the C-2 O-methyl group fits into a somewhat flexible antibody cavity and that hydrogen bonds involving the oxygen and, respectively, the OH at the 2- and 3-position of the sugar moiety as well as the 2'-position in the amide side chain are required. Monoclonal IgA ZAC-3 and IgG3 I-24-2 are specific for V. cholerae O:1 serotypes Ogawa/Inaba-LPS.1 The former did not show binding with members of either series of the synthetic ligands related to the O-antigens of the Ogawa or Inaba serotypes, in agreement with its reported specificity for the lipid/core region (1). Inhibition studies revealed that the binding of purified IgG3 I-24-2 to Ogawa-LPS might be mediated by a region in the junction of the OPS to the lipid-core region of the LPS. cDNA cloning and analysis of the anti-Ogawa antibodies S-20-4, A-20-6, and 2D6 revealed a very high degree of homology among the heavy chains. Among the light chains, no such homology between S-20-4 and A-20-6 on the one hand, and 2D6 on the other hand, exists. For the anti-Inaba/Ogawa antibodies I-24-2 and ZAC-3, their heavy chains are completely different, with some homology among the light chains.

Can repeated plasma donation by asymptomatic HIV-infected individuals delay the onset of AIDS?

Healthy HIV-positive regular donors of plasma in a programme of passive immunotherapy for AIDS patients were studied over a period of about two years. None developed symptoms of clinical progression; most seemed to make substantial gains of CD4 cells by comparison with asymptomatic individuals who were not donating. The effects of donation did not seem to diminish with repetition, and donor CD4 counts tended towards stabilizing within normal limits. Asymptomatic HIV-positive individuals were compared immunologically with 'normals' and people with AIDS, using a battery of 25 measurements on peripheral blood. The immunological profiles of donor and non-donor asymptomatics, indistinguishable at the start, became dissimilar: donors' profiles resembled AIDS less, non-donors became less like 'normal' and a few non-donor results could not be distinguished from AIDS. Improvement in the CD4 counts and amelioration of the immunological profile in donors provide prima facie evidence that plasmapheresis may be therapeutic for asymptomatic HIV-positive people. Further studies are justified.

Enzyme immunoassays using bispecific diabodies.

BACKGROUND: Bispecific antibodies with a first binding specificity to a target antigen and a second to an enzyme have great potential in enzyme immunoassays. As bispecific antibodies are difficult to make, the use of recombinant bispecific antibody fragments may provide a breakthrough. OBJECTIVES: To make bispecific antibody fragments directed against an enzyme and to demonstrate their application in enzyme immunoassays. STUDY DESIGN: Bispecific antibody fragments were assembled as diabodies (Holliger P., Prospero T., Winter G. Proc. Natl. Acad. Sci. USA 90, 1993, 6444-6448) directed to an enzyme, E. coli beta-galactosidase, and to each of three target antigens, hen-egg lysozyme (HEL), carcinoembryonic antigen (CEA), and HIV gpl20 (HIV). The diabodies were then evaluated in immunoassays. RESULTS: The HEL diabody was shown to recruit beta-galactosidase in a microtiter plate immunoassay in which diabody and enzyme were co-incubated with antigen, washed and enzyme substrate added. The CEA diabody was shown to detect CEA by immunocytochemical staining of transfected, CEA-expressing HeLa cells and of adenocarcinoma colon tissue sections, and the HIV diabody to detect gpl20 in immunoblots of total cell extracts. CONCLUSION: The results illustrate the diagnostic potential of diabodies in enzyme immunoassays.

Comparison of concentrations of Cry j 1 and Cry j 2 in diploid and triploid Japanese cedar (Cryptomeria japonica) pollen extracts.

Japanese cedar (Cryptomeria japonica) possibilities has been a serious allergic disease in Japan. There are two kinds of Japanese cedar trees; the popular one is diploid, the less popular is triploid. These trees are not very different morphologically. However, the relative allergenicity of their pollens is unknown, although both major allergens, Cry j 1 and Cry j 22, have been purified and cloned from the diploid line. Triploid trees are sterile and the allergenicity of their pollen may differ. Using Japanese-cedar-allergic patient sera, we compared the concentration of these two major allergens in both kinds of pollen. Pollen collected from different years and regions was also studied. The results indicate that triploid tree pollen extract has lower concentrations of both major allergens; therefore, the pollen may be less allergenic.

Characterization of a novel murine leukemia virus-related subgroup within mammals.

The murine leukemia virus (MuLV)-related retroviruses are one of seven genera which together constitute the family Retroviridae. They are widespread as both endogenous and exogenous agents within vertebrates and have been associated with a variety of malignancies and other disorders. We isolated and characterized 12 endogenous representatives of this genus from a number of mammalian hosts. Subsequent sequence analysis revealed that the isolated viruses cluster into two clearly distinct groups. All of the exogenous MuLV-related retroviruses which have been isolated to date, as well as several endogenous examples, fall into the first group, whereas the second group is represented solely by endogenous representatives, including human endogenous retrovirus type E (HERV.E). The two groups are widespread within mammals, with both often present within one animal species. Despite this, there is no evidence to date that recombination between members of the different groups has occurred. Genetic distances and several other properties of the HERV.E genome suggest that if exogenous members of this subgroup exist, they are likely to have biological properties different from those of the other exogenous viruses of this genus. Several of these viruses are known to have been integrated within their hosts' genomes for a long period of time, and a most recent divergence date for the MuLV and HERV.E subgroups can thus be proposed. This date, approximately 30 million years ago, is the most recent date possible, and it is probable that the actual period of time since their divergence is significantly longer.

Of four murine, anti-Shigella dysenteriae type 1 O-polysaccharide antibodies, three employ V-genes that differ extensively from those of the fourth.

Three murine, monoclonal antibodies, IgM 5286 F2, IgM 5297 C1, and IgG 5338 H4 were generated against Shigella dysenteriae type 1 O-specific polysaccharide (O-SP)-conjugate. They are specific for the O-SP, which is a poly-[alpha-L-rhamnopyranosyl-(1-->3)-alpha-L-rhamnopyranosyl-(1-->2)-al pha-D-galactopyranosyl-(1-->3)-2-deoxy-2-amino-N-acetyl-alpha-D-glucopyr anosyl]. The VH and VL genes of these antibodies were cloned and their sequences determined. They showed 93% homology, but were quite different to the primary sequence of IgM 3707 E9, of the same O-SP-specificity, previously reported. The fine-specificities of both IgG 5338 H4 and IgM 3707 E9 were for the same disaccharide moiety in the O-SP, while IgMs 5286 F2 and 5297 C1 showed fine-specificity for the entire repeating unit of the O-SP. Therefore, divergent sequences can confer upon antibodies similar-, or even identical-carbohydrate-epitope fine-specificity. In addition, close primary sequence-homology does not preclude differences in antibody fine-specificity.