Establishment and cryptic transmission of Zika virus in Brazil and the Americas.

Transmission of Zika virus (ZIKV) in the Americas was first confirmed in May 2015 in northeast Brazil. Brazil has had the highest number of reported ZIKV cases worldwide (more than 200,000 by 24 December 2016) and the most cases associated with microcephaly and other birth defects (2,366 confirmed by 31 December 2016). Since the initial detection of ZIKV in Brazil, more than 45 countries in the Americas have reported local ZIKV transmission, with 24 of these reporting severe ZIKV-associated disease. However, the origin and epidemic history of ZIKV in Brazil and the Americas remain poorly understood, despite the value of this information for interpreting observed trends in reported microcephaly. Here we address this issue by generating 54 complete or partial ZIKV genomes, mostly from Brazil, and reporting data generated by a mobile genomics laboratory that travelled across northeast Brazil in 2016. One sequence represents the earliest confirmed ZIKV infection in Brazil. Analyses of viral genomes with ecological and epidemiological data yield an estimate that ZIKV was present in northeast Brazil by February 2014 and is likely to have disseminated from there, nationally and internationally, before the first detection of ZIKV in the Americas. Estimated dates for the international spread of ZIKV from Brazil indicate the duration of pre-detection cryptic transmission in recipient regions. The role of northeast Brazil in the establishment of ZIKV in the Americas is further supported by geographic analysis of ZIKV transmission potential and by estimates of the basic reproduction number of the virus.

Immunosuppressive factor(s) extracted from lymphoid cells of nonresponder mice primed with L-glutamic acid60-L-alanine30-L-tyrosine10 (GAT) III. Immunochemical properties of the GAT-specific suppressive factor.

The GAT-specific suppressor T-cell factor (GAT-TsF) extracted from lymphoid cells from GAT-primed, nonresponder DBA/1 mice has been partially characterized. It is a protein that has affinity for GAT and determinants encoded by the I region of the H-2 complex. On the basis of specificity and avidity, GAT-TsF resembles anti-GAT-MBSA antibodies produced by DBA/1 mice in spite of the fact that it is too small to be classical antibody and has no constant-region determinants of heavy or light chains. Further, GAT or a fragment of GAT is associated with the GAT-TsF. GAT-TsF has been partially purified from the crude extract by absorption to GAT-Sepharose and elution with 0.4 to 0.6 KCl. GAT-TsF purified on the basis of its affinity for GAT bears I-region determinants but not detectable GAT or GAT fragment.

Restriction of primary responses to the IgG class and dependency of IgM responses on secondary immunization for the copolymers of L-glutamic acid, L-tyrosine, and L-alanine.

Primary responses to the linear polymers of L-glutamic acid, L-tyrosine, and L-alanine are restricted to the IgG class of antibodies. The appearance of specific IgM antibodies against these antigens is dependent upon secondary immunization, in contrast to many classical antigenic systems. The presence of an IgM response was verified by a direct plaque-forming cell assay, the inhibition of direct plaques by an antiserum specific for mouse micron-chain, and the physical separation of IgM and IgG GAT-specific antibodies by gel filtration. Preimmunization of the appropriate nonresponder strain with GAT or GT inhibits both the secondary IgM and IgG responses to GAT-MBSA and GT-MBSA, respectively. The tolerance observed is due to the induction of suppressor cells as demonstrated by cell transfer experiments.

Immunosuppressive factor(s) specific for L-glutamic acid50-L-tyrosine50 (GT) II. Presence of I-J determinants on the GT-suppressive factor.

The responses to the synthetic antigens, L-glutamic acid(60)-L- alanine(30)-L-tyrosine(10) (GAT) and L-glutamic acid(50)-L-tyrosine(50) (GT) are controlled by genes in the I region of the mouse H-2 complex (1-3). Preimmunization of the mice bearing the H-2(p,q,s) nonresponder haplotypes with GAT stimulates the development of suppressor T cells that inhibit in vivo or in vitro antibody responses to GAT complexed to the immunogenic carrier, methylated bovine serum albumin (GAT-MBSA) (4). The copolymer GT is not immunogenic in any inbred mouse strain tested, and has a suppressive effect on the antibody responses to GT-MBSA in mouse strains bearing the H-2(d,f,k,s) haplotypes; suppressor T cells have been demonstrated to be responsible for specific GT suppression (3). We have obtained specific suppressive extracts from thymus and spleen cells of GAT-or GT-primed suppressor strains (5,6). The specific suppressive T-cell factors in the active extracts have been characterized (6,7) and appear similar to the carrier-specific suppressor factor described by Tada and Taniguchi (8). These products belong to a family of newly identified molecules coded for by the I region of the H-2 complex with affinity for antigen and helper (9,10) or suppressive (5-8) regulatory activity on the immune response. Recently, Tada et al. have reported that the keyhole limpet hemocyanin (KLH)-specific suppressor factor is coded for by the I-J subregion of the H-2 complex (11). We now demonstrate also that a GT-specific suppressor factor extracted from the spleens and thymuses of B10.BR (H-2(k)) mice bears determinants controlled by the I-J subregion of the H-2 complex.

Immunosuppressive factor(s) specific for L-glutamic acid50-L-tyrosine50 (GT). III. Generation of suppressor T cells by a suppressive extract derived from GT-primed lymphoid cells.

Injection of mice with L-glutamic acid50-L-tyrosine50 (GT)- or L-glutamic acid60-L-alanine30-L-tyrosine10 (GAT)-specific suppressor T-cell factor (GT-TsF or GAT-TsF) up to 5 wk before antigenic challenge challenge suppresses GT-methylated bovine serum albumin (MBSA) and GAT-MBSA plaque-forming cells responses. T suppressor cells are responsible for the suppression induced by the suppressive extract as demonstrated by adoptive transfer and sensitivity to anti-Thy-1 and complement treatment. We conclude that suppressive extract induces specific suppressor T cells. The material responsible for generation of suppressor T cells is a product of the I subregion of the H-2 complex. We have excluded that suppressive quantities of antigens are present in the extract. A/J mice, which can neither be suppressed by GT nor make GT-TsF can be suppressed by BALB/c GT-tsf. Spleen cells from BALB/c GT TsF-primed A/J mice can adoptively transfer suppression to normal syngeneic recipients. A/J mice appear to be genetically defective in cells involved in factor production. These results are discussed in the light of a two-step model for induction of antigen-specific suppressor cells.

Antigen-specific T-cell-mediated suppression. I. Induction of L-glutamic acid60-L-alanine30-L-tyrosine10 specific suppressor T cells in vitro requires both antigen-specific T-cell-suppressor factor and antigen.

A combination of in vitro and in vivo techniques were used to explore the mode of action of both crude and purified suppressive extracts specific for the random copolymer L-giutamic acid(60)-L-alanine(30)-L-tyrosine(10) (GAT- T(s)F) obtained from nonresponder DBA/1 (H-2(q)) mice. Normal DBA/1 spleen cells were incubated under modified Mishell-Dutton culture conditions for 2 days together with crude or purified GAT-T(s)F, and in the presence or absence of free GAT. These cells were then washed extensively and 3 x 10(6) viable cells transferred to syngeneic recipients, which were challenged at the same time with the immunogenic form of GAT complexed to methylated bovine serum albumin (GAT-MBSA). GAT-specific IgG plaque-forming cells (PFC) in the spleen were assayed 7 days later. In agreement with earlier in vitro studies on the action of GAT-T(s)F, it was demonstrated that under these conditions, low concentrations of GAT-T(s)F stimulated the development of cells which, aider transfer, are able to suppress the GAT PFC response to GAT-MBSA. The cells responsible for this suppression were shown to be T lymphocytes by using nylon wool-purified T cells for suppressor cell induction and by eliminating suppressive activity in cells cultured with crude GAT-T(s)F by treatment with anti-Thy 1.2 plus C before transfer. The suppressor T cells act in a specific manner failing to suppress significantly either anti-sheep erythrocyte or trinitrophenyl-ovalbumin primary PFC responses. For the induction of GAT-specific suppressor T cells in culture, a moiety bearing H- 2(K(q) or I(q)) determinants and also GAT, either bound to the crude GAT- T(s)F or added in nanogram amounts to antigen (GAT)-free purified GAT-T(s)F, were both required.

The first alpha helix of interleukin (IL)-2 folds as a homotetramer, acts as an agonist of the IL-2 receptor beta chain, and induces lymphokine-activated killer cells.

Interleukin (IL)-2 interacts with two types of functional receptors (IL-2Ralphabetagamma and IL-2Rbetagamma) and acts on a broad range of target cells involved in inflammatory reactions and immune responses. For the first time, we show that a chemically synthesized fragment of the IL-2 sequence can fold into a molecule mimicking the quaternary structure of a hemopoietin. Indeed, peptide p1-30 (containing amino acids 1-30, covering the entire alpha helix A of IL-2) spontaneously folds into an alpha-helical homotetramer and stimulates the growth of T cell lines expressing human IL-2Rbeta, whereas shorter versions of the peptide lack helical structure and are inactive. We also demonstrate that this neocytokine interacts with a previously undescribed dimeric form of IL-2Rbeta. In agreement with its binding to IL-2Rbeta, p1-30 activates Shc and p56(lck) but unlike IL-2, fails to activate Janus kinase (Jak)1, Jak3, and signal transducer and activator of transcription 5 (STAT5). Unexpectedly, we also show that p1-30 activates Tyk2, thus suggesting that IL-2Rbeta may bind to different Jaks depending on its oligomerization. At the cellular level, p1-30 induces lymphokine-activated killer (LAK) cells and preferentially activates CD8(low) lymphocytes and natural killer cells, which constitutively express IL-2Rbeta. A significant interferon gamma production is also detected after p1-30 stimulation. A mutant form of p1-30 (Asp20-->Lys), which is likely unable to induce vascular leak syndrome, remains capable of generating LAK cells, like the original p1-30 peptide. Altogether, our data suggest that p1-30 has therapeutic potential.

Genomic and epidemiological monitoring of yellow fever virus transmission potential.

The yellow fever virus (YFV) epidemic in Brazil is the largest in decades. The recent discovery of YFV in Brazilian Aedes species mosquitos highlights a need to monitor the risk of reestablishment of urban YFV transmission in the Americas. We use a suite of epidemiological, spatial, and genomic approaches to characterize YFV transmission. We show that the age and sex distribution of human cases is characteristic of sylvatic transmission. Analysis of YFV cases combined with genomes generated locally reveals an early phase of sylvatic YFV transmission and spatial expansion toward previously YFV-free areas, followed by a rise in viral spillover to humans in late 2016. Our results establish a framework for monitoring YFV transmission in real time that will contribute to a global strategy to eliminate future YFV epidemics.

First Complete Genome Sequences of Zika Virus Isolated from Febrile Patient Sera in Ecuador.

Here, we present the complete genome sequences of two Zika virus (ZIKV) strains, EcEs062_16 and EcEs089_16, isolated from the sera of febrile patients in Esmeraldas City, in the northern coastal province of Esmeraldas, Ecuador, in April 2016. These are the first complete ZIKV genomes to be reported from Ecuador.

Functional and physical association of a cell surface phospholipid and interleukin-2 receptor p55(alpha) subunits.

A phosphatidylcholine-like phospholipid expressed in the outer leaflet of the cell membrane shortly after mitogenic activation of T-cells is described, based on the binding of monoclonal antibody 90. 60.3. Expression of the 90.60.3 phospholipid antigen in T-cells is activation-dependent. Once expressed, the 90.60.3 phospholipid is in direct physical association with the interleukin-2 (IL-2) binding domain of IL-2 receptor alpha subunits, but does not affect IL-2 binding. The association is specific, because the 90.60.3 phospholipid is not found in association with other domains of IL-2 receptor alpha subunits, or near IL-2 receptor beta or gamma subunits. Culturing cytokine-dependent cell lines in the presence of monoclonal antibody 90.60.3 potentiates IL-2-dependent cell survival and proliferation in a dose-dependent manner. In contrast, IL-4-dependent responses are not potentiated. Taken together, the data suggest that specific plasma membrane phospholipids expressed in the outer leaflet after T-cell activation associate with the IL-2 binding domain of IL-2 receptor alpha subunits (and perhaps other cytokine receptors), and may play a role in regulating receptor mobility or signal transduction.

Characterization of three monoclonal antibodies specifically directed against the ligand binding site area of the p55 subunit of the mouse interleukin-2 receptor.

Three new rat monoclonal antibodies (MAbs) (5A2, 125A8 and 135D5) directed against the mouse interleukin-2 receptor (IL-2R) were isolated. They were obtained after immunization of LOU rats with 14.1.6 T helper cell clones. These three MAbs recognize the p55 subunit of the IL-2R and compete with the binding of previously characterized MAbs AMT13 and 3C7 specific for this p55 subunit [Moreau et al. (1987) Eur. J. Immun. 15, 723-727]. They recognize the same (or closely related epitopes) since they reciprocally compete with each other's binding. Scatchard plot analysis of the data from inhibition experiments clearly indicate that they recognize with very high affinity the ligand binding site area of the p55 subunit of the IL-2R. The properties of the Fab fragment prepared from 5A2 and 135D5 indicate that at saturation one intact IgG molecule binds two IL-2R molecules.

Induction of mouse p55 interleukin-2 receptor gene expression by IL-2 and IL-4 and characterization of its transcription initiation sites.

Two murine T cell lines (C30.1 and Line 1) were used to study the expression of the p55 interleukin-2 receptor gene. C30.1 is an IL-2-dependent T cell line that can be stimulated for a short period of time by IL-4. Line 1 cells are propagated in IL-4 but they also proliferate in response to IL-2. In both cell lines stimulation by IL-2 leads to a strong induction of p55 IL-2 receptor mRNA while stimulation by IL-4 leads only to a very moderate increase in expression of this mRNA. The induction of p55 IL-2 receptor mRNA by IL-4 is comparable to that of beta-actin mRNA. These data confirm that IL-2 upregulates p55 IL-2 receptor gene expression while IL-4, which also activates T cells, does not lead to specific induction of this gene. We have also determined the transcription initiation sites utilized by the p55 IL-2 receptor gene in C30.1 and Line 1 cells. Seven sites were identified, one of which predominates. Resting cells, or cells stimulated with IL-2 or IL-4, display the same pattern of transcription site utilization.

Single peptide and anti-idiotype based immunizations can broaden the antibody response against the variable V3 domain of HIV-1 in mice.

The third variable (V3) domain of the human immunodeficiency virus type 1 (HIV-1) external envelope glycoprotein gp120 is a major target of neutralizing antibodies in infected persons and in experimental immunized animals. Given the high degree of sequence variability of V3, the humoral response toward this region is very type-specific. In the present study, we evaluated the potential of a single peptide and an anti-idiotypic antibody to broaden the anti-V3 antibody specificity in BALB/c mice. We show that a synthetic peptide derived from the V3 determinant of HIV-1 MN isolate (V3MN), when used as an immunogen, was able to induce an antibody response to multiple (up to six) HIV-1 strains. The extent of this cross-reactivity, which tended to enlarge as the injections increased, appeared to be inversely correlated with the binding affinity to V3MN peptide. These data thus present evidence that, despite its great sequence heterogeneity, the V3 loop encompasses conserved amino-acid positions and/or stretches which may be less immunogenic than their variable counterparts. We additionally demonstrate that a rabbit anti-idiotype (Ab2), recognizing a binding site related idiotype on a V3-specific mouse monoclonal antibody (Ab1), could mount a broadened humoral response (Ab3) in mice. Unlike nominal antibody Ab1 which strictly reacted with the European HIV-1 LAI isolate, elicited Ab3 recognized the two divergent HIV-1 strains SF2 and 1286, originating respectively from North America and Central Africa, in addition to LAI. The reasons accounting for this Ab2-induced enlargement of the V3 antibody response are discussed. Our findings suggest that single peptide and anti-idiotype based immunizations may provide viable approaches to overcome, at least in part, HIV epitope variability.

Monoclonal anti-GAT antibodies with different fine specificities express the same public idiotype.

We have studied the idiotypic specificities expressed by 22 anti-GAT hybridoma products (HP). These antibodies, although derived from cells of mice with three distinct heavy-chain linkage groups (BALB/c, Igh-1a, DBA/2, Igh-1c and C57BL/6, Igh-1b) all express the same public idiotypic specificity, p. GAT, defined by the heterologous binding of anti-idiotypic serum 715 to C57BL/6 anti-GAT antibodies. None of these antibodies expressed the strain-restricted idiotypic specificity, s.r. GAT-1, defined by the binding of anti-idiotypic serum JL 122 to BALB/c anti-GAT antibodies. BALB/c anti-GAT HP could be shown to fall into three subsets with respect to their fine antigenic specificity for GAT, GT and GA. An individual idiotypic specificity, i1-GAT (defined by syngeneic anti-idiotypic sera raised against one of the BALB/c HP), was also found on a group of BALB/c HP which all shared a similar fine antigenic specificity pattern. Taken together, these observations suggest that the expressed mouse anti-GAT repertoire derives from a very few V-germ-line genes (VH-GAT and VK-GAT) which are highly conserved in the species, and which determine the structure resulting in the p. GAT idiotypic specificity. The variations in fine specificity and individual idiotype are likely therefore to reflect somatic variations affecting these germ-line genes.

Characterization of monoclonal antibodies against prostaglandin E2: fine specificity and neutralization of biological effects.

The specificity and heterogeneity of the immune response of BALB/c mice immunized with prostaglandin E2 (PGE2) coupled to thyroglobulin was studied. All the animals (n = 50) responded to PGB2, a transformation product of PGE2. However, following repeated injections most of the animals (n = 30) were also able to respond to PGE2. Cellular hybridizations were performed and five anti-PGE2 monoclonal antibodies were isolated and analysed. They are mainly directed against the ring and the omega-chain of PGE2 but their specificity toward the alpha-chain is more limited. The association constants are greater than to 1 X 10(9) M-1. The monoclonal antibody 8E.57.71 (Ka = 1.3 X 10(10) M-1) is particularly convenient for sensitive radioimmunoassays (detection limit 25pg/ml, when iodinated tracer is used). Anti-PGE2 monoclonal antibodies were found to neutralize the specific binding of [3H]PGE2 to rat brain hypothalamic receptors and to inhibit the PGE2 induction of rat fundus muscular contraction.

Immunochemical characterization of antigenic domains on human IL-2: spatially distinct epitopes are associated with binding to the p55 and p70 subunits of IL-2 receptor.

We have isolated and characterized 8 mAb against human rIL-2. All recognize nonglycosylated rIL-2 in liquid phase with similar affinities (Kd approximately 1 nM). Based on the epitopes of the IL-2 molecule that they recognize and their pattern of reactivity against glycosylated and non-glycosylated IL-2, they have been classified into four groups. The first group of anti-IL-2 mAb (2C4, 19B11 and 12C2) inhibits IL-2 binding to p70 IL-2R, while the second one (16F11, 18E1 and 2A4) prevents its binding to p55 IL-2R. These two groups neutralize IL-2 activity in a T cell proliferation assay equally well, due to their similar inhibition of IL-2 binding to high affinity IL-2R. Two mAb, 3H9 and 17F4, recognize separate epitopes on IL-2 molecule, are poor inhibitors of IL-2 binding, and they are inefficient in the neutralization of its biological activity; they have been assigned to the third and fourth groups, respectively. These results show that mAb from the first and second group recognize two epitopes of the human IL-2 molecule which probably overlap the p70 IL-2R and p55 IL-2R binding sites, respectively. In addition, these areas together form the high affinity IL-2R binding site. The two mAb from the third and fourth group recognized epitopes of IL-2 not directly involved in IL-2 binding to its receptor. All eight mAb anti-human IL-2 recognize murine IL-2 and with the exception of one, 17F4 mAb are also able to neutralize it in a T cell proliferation assay. The relationship between the structure and the function of the IL-2 molecule is discussed.

Characterization of a monoclonal antibody directed against the NH2 terminal area of interleukin-2 (IL-2) and inhibiting specifically the binding of IL-2 to IL-2 receptor beta chain (IL-2R beta).

An anti-human IL-2 mAb (19B11/beta) was found to selectively block the binding of IL-2 to TS1 beta cells expressing the interleukin-2 receptor beta (IL-2R beta) without affecting binding to TS1 alpha cells expressing the IL-2R alpha receptor. It also specifically inhibits the IL-2 driven cell proliferation in TS1 beta cells. These observations have lead to the hypothesis that its epitope is related to an IL-2 area involved in binding with IL-2R beta chain. This epitope was identified using various peptides covering the N-terminal half (including alpha helix A) of the 133 amino acids of IL-2. MAb 19B11/beta does not recognize peptides 30-54 and 44-54 but recognizes peptides 1-22 and 1-30 with a good affinity. Furthermore, threonine in position no. 3 was found to be critical for the binding of mAb 19B11/beta. A relationship between the epitope of mAb 19B11/beta and the glycosylation of the IL-2 molecule was observed. This further demonstrates that the NH2 terminal area of IL-2 is critical for IL-2/IL-2R beta interactions. Two other mAbs were studied during the course of this work. They served as control for the study of mAb 19B11/beta and provide some additional insight concerning the question of IL-2/IL-2R structure-function. MAb 16F11/alpha selectively blocks the IL-2 binding to TS1 alpha cells. The epitope of mAb 16F11 is conformational and it was not possible to study the corresponding IL-2/IL-2R alpha region of interaction. Epitope of mAb 3H9 is localized between residues 30 and 54 and does not affect the binding of IL-2 to IL-2R alpha.

Analysis of a major rat idiotype associated with Anti-GAT antibodies.

An anti-idiotypic antiserum was raised in a rabbit against a pool of purified F.344 rat anti-GAT antibodies. GAT-13, the idiotype defined by this serum, is present in all F.344 anti-GAT sera from primary and secondary anti-GAT responses. Anti-GAT sera of 13 inbred rat strains, with different RT1 haplotypes and with different heavy- and light-chain allotypes, all express idiotypic determinants cross-reacting with GAT-13. Thus, like in mice anti-GAT antibodies from rats express public idiotypic determinants. The anti-idiotypic serum also recognizes a highly conserved idiotypic specificity present on mouse and guinea-pig anti-GAT antibodies. The mouse, rat and guinea-pig express a similar highly conserved idiotypic specificity after immunization with GAT. All anti-GAT antibodies from the mouse and guinea-pig bear this idiotypic specificity. These results confirm the existence in the anti-GAT response of interspecies cross-reactive idiotypic determinants.

Covalent linkage of the synthetic adjuvant MDP to the synthetic polypeptide (T,G)-A-L changes the specificity of the immune response at the T and B cell level.

It is now well established that the synthetic molecule MDP (N-acetylmuramyl-L-alanyl-D-isoglutamine) can be a good adjuvant of immunity when covalently linked to antigen. The question raised in this work is whether conjugation of antigen to the immunomodulatory molecule MDP can modify the specificity of the antibodies and T cells induced following immunization. Using the well characterized synthetic polypeptide antigen, poly(L-Tyr,L-Glu)-poly(DL-Ala)--poly(L-Lys) [(T,G)-A--L], we show that immunization of C57B1/6 (H-2b) mice with MDP-(T,G)-A--L conjugate elicits at least two types of antibody directed against the poly(DL-Ala)--poly(L-Lys) (A--L) part of the antigen, and against new determinant(s) formed by MDP and a portion of the (T,G)-A--L molecule. Interestingly, the poly-(L-Try L-Glu) side chains thought to constitute the major antigenic determinants of the (T,G)-A--L molecule were not recognized. Lymph node cells from (T,G)-A--L immunized mice can be equally well stimulated in vitro by (T,G)-A--L or by MDP-(T,G)-A--L, whereas lymph node cells from MDP-(T,G)-A--L primed animals can be stimulated only when challenged by the conjugate used for immunization, and not by the free synthetic polypeptide (T,G)-A--L. The data presented here show that the coupling of a low mol. wt molecule such as MDP (mol. wt approx. 500) to an antigen can greatly modify the immune response directed against this antigen. Furthermore, (1) different antibody specificities are elicited depending upon whether the priming is done with free MDP and antigen or with MDP covalently linked to the antigen; (2) although still accessible on the conjugate, an epitope which represents the major antigenic determinant on the free polypeptide appears to be silent when presented on the conjugate; and (3) new determinant(s) formed by the chemical linkage of the polypeptide to the synthetic adjuvant are involved in the priming of T cells.

Contribution of the H- and L-chains and of the binding site to the idiotypic specificities of mouse anti-GAT antibodies.

The contribution of the H- and L-chains to the structure of the main idiotype of anti-poly (Glu60-Ala30-Tyr10) (GAT) antibodies has been studied. This idiotype has been previously divided into four types of specificity: (1) the highly conserved idiotypic specificity (h.c. GAT) is expressed by anti-GAT antibodies from the guinea-pig, rat and mice; (2) the public specificity (p. GAT) is expressed in an identical form by all anti-GAT antibodies from all strains of mice tested and by all hybridoma products (HP) with anti-GAT activity; (3) the strain-restricted specificity (s.r. GAT-1) is only expressed by anti-GAT antibodies from strains with Ig-1a, Ig-1c and Ig-1c allotypic markers; and finally (4) the individual specificity i1-GAT defined on HP G5 is also expressed by most of the hybridoma protein with anti-poly (Glu50-Tyr50) (GT) activity. In this paper we show that h.c.GAT, p.GAT and i1-GAT require the interaction of H- and L-chains to be expressed: (1) isolated H- and L-chains from HP G5 did not express these specificities; and (2) recombinant molecules composed of H- and L-chains from HP with anti-GAT activity and an irrelevant myeloma protein (MOPC21) never expressed h.c.GAT, p.GAT and i1-GAT. We next investigated the relationship between the GAT binding site and the p.GAT, h.c.GAT and s.r.GAT-1 idiotypic specificities. GAT and GT were not able to inhibit the binding to s.r.GAT-1 while they inhibit the idiotypic binding to p.GAT and h.c.GAT. A GAT fragment of mol. wt 3000 was also shown to inhibit the binding of p.GAT and h.c.GAT to the appropriate sera. Thus p.GAT and h.c.GAT are very close to the GAT combining site while s.r.GAT-1 represents an idiotypic specificity located outside the GAT binding site.