Protection of macaques against vaginal transmission of a pathogenic HIV-1/SIV chimeric virus by passive infusion of neutralizing antibodies.

The development of the human immunodeficiency virus-1 (HIV-1)/simian immunodeficiency virus (SIV) chimeric virus macaque model (SHIV) permits the in vivo evaluation of anti-HIV-1 envelope glycoprotein immune responses. Using this model, others, and we have shown that passively infused antibody can protect against an intravenous challenge. However, HIV-1 is most often transmitted across mucosal surfaces and the intravenous challenge model may not accurately predict the role of antibody in protection against mucosal exposure. After controlling the macaque estrous cycle with progesterone, anti-HIV-1 neutralizing monoclonal antibodies 2F5 and 2G12, and HIV immune globulin were tested. Whereas all five control monkeys displayed high plasma viremia and rapid CD4 cell decline, 14 antibody-treated macaques were either completely protected against infection or against pathogenic manifestations of SHIV-infection. Infusion of all three antibodies together provided the greatest amount of protection, but a single monoclonal antibody, with modest virus neutralizing activity, was also protective. Compared with our previous intravenous challenge study with the same virus and antibodies, the data indicated that greater protection was achieved after vaginal challenge. This study demonstrates that antibodies can affect transmission and subsequent disease course after vaginal SHIV-challenge; the data begin to define the type of antibody response that could play a role in protection against mucosal transmission of HIV-1.

Protection of rhesus macaques against disease progression from pathogenic SHIV-89.6PD by vaccination with phage-displayed HIV-1 epitopes.

The antigenic polymorphism of HIV-1 is a major obstacle in developing an effective vaccine. Accordingly, we screened random peptide libraries (RPLs) displayed on phage with antibodies from HIV-infected individuals and identified an array of HIV-specific epitopes that behave as antigenic mimics of conformational epitopes of gp120 and gp41 proteins. We report that the selected epitopes are shared by a collection of HIV-1 isolates of clades A-F. The phage-borne epitopes are immunogenic in rhesus macaques, where they elicit envelope-specific antibody responses. Upon intravenous challenge with 60 MID50 of pathogenic SHIV-89.6PD, all monkeys became infected; however, in contrast to the naive and mock-immunized monkeys, four of five mimotope-immunized monkeys experienced lower levels of peak viremia, followed by viral set points of undetectable or transient levels of viremia and a mild decline of CD4+ T cells, and were protected from progression to AIDS-like illness. These results provide a new approach to the design of broadly protective HIV-1 vaccines.

V3 seroreactivity and sequence variation: tracking the emergence of V3 genotypic variation in HIV-1-infected patients.

OBJECTIVE: To investigate the relationship between V3-specific immune responses and viral quasispecies evolution in 10 HIV-1-seropositive patients enrolled in a phase I trial of recombinant gp160. METHODS: Serologic responses to the HIVLAI V3 loop and autologous V3 loop DNA sequences were sequentially determined over a 3-4-year interval. RESULTS: Six patients either seroconverted or had a > or = 42-fold boost in titer to the V3 reagent associated with an average of 3.2 amino-acid changes in their autologous V3 loops. Four patients with < or = 11-fold change in titer to the V3 loop showed an average of 0.75 amino-acid changes. Attempts to measure autologous V3 loop responses in four patients using a peptide enzyme-linked immunosorbent assay technique did not show a distinct binding preference for autologous versus heterologous V3 loop peptides. Thus, we interpret seroreactivity to the heterologous HIVLAI V3 loop to reflect the broadness of the V3 immune response rather than a direct measure of epitope-specific immune pressure. CONCLUSIONS: These data suggest that the broadness of serologic responses to viral epitopes are reflected in the rate of evolution of their cognate coding sequences and support the view that the immune response to HIV-1 results in the continuous selection of new viral variants during the course of disease.

Presentation of HIV V3 loop epitopes for enhanced antigenicity, immunogenicity and diagnostic potential.

OBJECTIVE: To evaluate the immunological properties of a panel of human mucin MUC1/HIV V3 loop chimeras. DESIGN: The immunodominant epitope of MUC1 (APDTR) was found to be structurally isomorphous with the tip of the principle neutralizing determinant (PND) of HIV-1 (MN) (GPGRA). A panel of 120 residue, six tandem repeat (TR) and 60 residue, three TR chimeric antigens were constructed in which the repeating MUC1 epitope is replaced by HIV-1 PND. Each 20 residue TR contains one PND epitope. The PND of HIV-1 is presented in the native beta-turn conformation at the crest of each repeating knob structure of the mucin-like protein. METHODS: The antigenicity of the chimeric antigens were compared using enzyme-linked immunosorbent assay (ELISA) and HIV-infected patient sera. Structural effects of antibody-antigen interactions were determined using surface plasmon resonance, with human monoclonal antibodies, chimeric antigens and the cyclic and linear V3 loops. Immunogenicity of three versus six TR was measured in mice. RESULTS: Nine residues of the HIV PND substituted into the mucin backbone were equivalent to the 36 residue cyclic V3 loop in ELISA. The 120 residue antigens induced high titer, immunoglobulin (Ig) M and IgG, and HIV-specific antibodies in mice. CONCLUSIONS: MUC1/V3 chimeras efficiently detect HIV-specific antibodies in patient sera. Multivalent presentation of the PND is advantageous for higher affinity antibody-antigen interactions and for inducing HIV-specific IgM and IgG antibodies.

Measurement of cerebrospinal fluid antibody to the HIV-1 principal neutralizing determinant (V3 loop).

Antibody to the human immunodeficiency virus (HIV)-1 principal neutralizing determinant (V3 loop) was measured by peptide enzyme-linked immunosorbent assay (ELISA) in cerebrospinal fluid (CSF) and paired serum samples of 21 HIV-seropositive patients. These patients had normal neurologic examinations and were without neurologic symptoms. Peptide ELISA demonstrated intrathecal antibody synthesis against the V3 loop of HIVMN, the V3 loop of HIVNY5, the V3 loop of HIVLAI, and the entire recombinant HIV-1MN gp120 in 21 of 21, 10 of 21, one of 21, and 12 of 21 patients, respectively. Biospecific interaction analysis (BIAcore), which requires only small amounts of CSF, was also used to detect anti-V3 CSF antibody. Fine mapping of linear epitopes within the V3 region was successful in three of five patients by Geysen PIN (PEPSCAN) ELISA and discordance between epitope specificity of CSF and serum antibody was found. While detection of CSF antibody against the V3 loop of HIVMN by peptide ELISA has been recently reported, we add to this finding using the peptide ELISA, PEPSCAN and BIAcore methodologies as well as measuring intrathecal antibody synthesis against V3 loops from HIV strains. Application of these techniques to future studies of anti-V3 antibody in CSF from persons receiving anti-HIV-1 immunizations may provide insight into the immunoregulation of the virus in the nervous system.

Preferential antibody recognition of structurally distinct HIV-1 gp120 molecules.

We have developed an assay, using a biosensor matrix and surface plasmon resonance, that rapidly and reproducibly measures antibody reactivity to human immunodeficiency virus type 1 (HIV-1) gp120 in various structural conformations. In particular, antibodies displaying preferential reactivity to a CD4-binding competent ("native," rgp120) or CD4-binding incompetent ("reduced," rcmgp120) monomeric gp120 molecule were distinguished. This technique has advantages over conventional enzyme-linked immunosorbent assay (ELISA) methodology in which it is difficult to control the concentration of protein adsorbed to the ELISA wells and a significant disruption of protein structure occurs on adsorption. A population of gp120 molecules that lacked CD4 receptor binding capacity and bound antibodies specific for reduced gp120 was found in several native gp120 preparations. The relative amount of this CD4-binding incompetent population varied among the various preparations studied. This presence of CD4-binding incompetent molecules within various native recombinant gp120 preparations may have implications for HIV-1 envelope vaccine development. By measuring antibody-binding ratios, several monoclonal antibodies were identified, which, although elicited by immunization with various native gp120 preparations, bound specifically to reduced gp120. The ability to screen antibody specificity against HIV-1 envelope proteins with different conformations will assist in determining the quality of antibodies induced by various HIV-1 envelope vaccine candidates.

Real-time biospecific interaction analysis of antibody reactivity to peptides from the envelope glycoprotein, gp160, of HIV-1.

A new assay designed to quantitate antibody reactivity to specific peptides using biospecific interaction analysis (BIAcore) has been developed. Peptides of various lengths (15-40 amino acids) and isoelectric points (pI = 4.5-13) were covalently linked (immobilized) to a biosensor and interacted with polyclonal human sera. The immobilization procedure was highly reproducible, with bound peptides retaining high antibody reactivity. The assay was rapid, requiring only 25-30 min to immobilize the peptide and 2-8 min for each subsequent peptide/serum binding interaction. The same peptide surface has been used for up to 90 cycles of serum binding and regeneration with only a 0.3% decay in reactivity over cycle number. The quantitative BIAcore signal, measuring peptide/antibody binding interactions, was directly related to the antigen/antibody concentrations within the biosensor. The assay allowed interactants to be studied in their native form and without the need of additional secondary detection antibodies. Correlation between conventional peptide ELISA and BIAcore was obtained. The BIAcore linear range persisted over a series of eight two-fold dilutions. This extended linear dynamic response range is an improvement over conventional ELISA measurements. The sensitivity for monoclonal antibody detection is similar to conventional ELISAs and 4.9 ng/ml was readily detected.

Characterization of a soluble, oligomeric HIV-1 gp160 protein as a potential immunogen.

We have assessed the oligomeric structure and antigenic properties of an affinity purified gp160 protein (oligo-gp160) using biosensor technology. Sucrose gradient purification analysis identified the existence of tetrameric, dimeric and monomeric forms of the protein. Reactivity to a broad panel of monoclonal antibodies specific for oligomeric gp160, discontinuous epitopes within monomeric gp120 and several linear epitopes within gp120 (V3) and gp41 was demonstrated. International sera from several countries, where HIV-1 clades A-F are prevalent, including type O from Cameroon, were reactive with oligo-gp160 indicating conserved antigenic epitopes. Enhanced immunologic reactivity per gp160 molecule was obtained with oligo-gp160 as compared to other current HIV-1(IIIB) subunit monomeric envelope gp120/gp160 immunogens suggesting higher HIV-1 envelope protein mimicry. HIV-1 antibodies from sera during acute HIV-1 infection were detectable by oligo-gp160 prior to detection with either a recombinant, monomeric gp120 protein or several commercial HIV-1 screening kits suggesting antibodies sensitive to oligomeric gp160 structure may be present earlier in infection. The oligomeric nature of this gp160 protein preparation and high reactivity with divergent mAbs and HIV-1 sera support the use of this protein as an HIV-1 immunogen.

Differential role of V3-specific antibodies in neutralization assays involving primary and laboratory-adapted isolates of HIV type 1.

To identify epitopes important in neutralizing primary HIV-1 isolates, we have selectively depleted HIV-1 sera of antibodies specific for the third hypervariable region (V3) of the HIV-1 envelope glycoprotein gp120, and then assessed the functional consequences of such depletion in neutralization assays. The nucleotide sequence of the V3 loop region from HIV-1 PBMC DNA was determined for three HIV-1-infected patients, corresponding peptides were synthesized, and then subsequently used for V3 depletion of the patient sera. Depletion using a single clade B V3 peptide was capable of depleting > 98% of binding antibodies to multiple clade B V3 peptides, including those with changes within the GPGX tip of the loop. Depleted and undepleted sera were studied for their ability to neutralize both laboratory-adapted HIV-1MN and two primary HIV-1 isolates with known V3 sequences, using a viral infectivity reduction assay. While the majority of HIV-1MN neutralization was lost on V3 depletion, the loss in neutralization capacity against primary isolates by these same V3-depleted sera was substantially less pronounced. This suggests that V3 peptide-specific antibodies within HIV-1 serum play a fundamentally different role in mediating neutralization in assays involving laboratory-adapted and primary isolates and implicates antibodies with epitope specificities outside of V3 as major determinants in neutralization assays involving primary isolates.

A human monoclonal antibody specific for the V3 loop of HIV type 1 clade E cross-reacts with other HIV type 1 clades.

To ascertain the antigenic relationship between HIV-1 viruses belonging to various genetically defined subgroups (clades), shared epitopes need to be defined. Human monoclonal antibodies (MAbs) are particularly useful for this purpose because they can detect complex regions of viral proteins that may be missed by sequence analysis and because, by definition, they react with epitopes that stimulate the human immune system. Monoclonal antibodies derived from the cells of HIV-1 clade B-infected subjects have been used extensively for this purpose. Here we describe the first human MAb derived from a clade E-infected individual; the MAb is specific for the V3 loop, recognizing a core epitope represented by the amino acids TRTSVR on the N-terminal side of the crown of the V3 loop. The IgG1(kappa) MAb, designated 1324E, binds to the clade E consensus V3 loop, to rgp120 proteins from clade E and to peripheral blood mononuclear cells infected in vitro with the virus that infected the subject from whose cells the MAb-producing heterohybridoma was derived. Strong cross-reactivity of the MAb to the V3 peptides, rgp120 proteins, and native monomeric gp120s representing clades A and C, as well as to cells infected with a clade C primary isolate, revealed a shared V3 epitope between these clades. When tested for its neutralizing ability, MAb 1324E neutralized a clade E isolate that had been adapted for growth in H9 cells but failed to neutralize five clade E primary isolates.

HIV type 1 subtype E-infected patients with broadened, dual (B/E) V3 loop serology have increased cross-neutralizing antibodies.

The two prevalent subtypes of HIV-1 circulating in Thailand are subtypes E and B. While the most prevalent subtype continues to be E using molecular typing assays, immunologically, a subset of subtype E-infected patients (3.4% in 1997) have binding antibodies to both the E and B V3 loops in a peptide ELISA. To assess the potential function of this dual (B/E) V3 reactivity, plasmas from patients with genetically defined HIV-1 subtype E infection and either E or B/E V3 serotypes were compared for magnitude and breadth of neutralization of seven primary and laboratory-adapted subtype B and E viruses. Dually reactive (B/E) plasmas showed significantly increased cross-neutralizing activity against subtype B viruses (p < 0.001), and increased neutralization of the panel of viruses overall (p < 0.02), as compared to monoreactive E serotype plasmas. While the total envelope binding antibody titers to both subtype B and E envelopes did not differ significantly between the E and B/E plasmas, 67% of B/E plasmas neutralized >50% of the viruses in the panel, and only 14% of E plasmas showed this broadened neutralizing activity. These data suggest that dual (B/E) V3 loop reactivity may be a marker of broader immune recognition of HIV envelope epitopes in subtype E-infected patients. V3 loop antibody, perhaps in conjunction with antibodies to additional epitopes, may play a role in neutralization of virus isolates from Thailand.

HIV type 1 subtypes in Malaysia, determined with serologic assays: 1992-1996.

We investigated the molecular epidemiology of HIV-1 subtypes in Malaysia among injecting drug users (IDUs) and sexual transmission risk groups, using serologic and genetic techniques. Frozen sera collected at a general hospital, a blood bank, several drug treatment centers, and an STD clinic in Kuala Lumpur, between 1992 and 1996, were investigated retrospectively. V3 peptide serotyping and monomeric gp120 capture serotyping were used to study 89 known HIV-1-infected subjects. The methods differentiate subtypes B, E, and C. V3 peptide and gp120 capture results were comparable. No subtype C-specific reactive sera were found; one specimen was dually reactive for subtypes C and B, using the V3 peptide ELISA; and four were durally reactive for subtypes E and C using this assay. Genotypic analysis of HIV-1 gag RNA in serum was done on a subset of subjects and confirmed serologic findings. HIV-1 subtypes differed significantly by risk category: of 53 IDUs, 29 (55%) were infected with subtype B and 19 (36%) were infected with subtype E, 3 (6%) were dually reactive, and 2 (4%) were not typable. Of 36 persons with heterosexual risks, 29 (81%) were infected with subtype E, 5 (14%) were infected with subtype B, and 2 (5%) were not typable. Persons with IDU risks were significantly more likely to be infected with subtype B than were those with sexual risks (OR 5.89; 95% CI, 1.94-18.54; p < 0.001). Subtypes B and E of HIV-1 appear to predominate in Malaysia; subtype B was more prevalent among IDUs; subtype E was more prevalent among all other groups. These results may have important HIV-1 vaccine implications.

Macaque dendritic cells infected with SIV-recombinant canarypox ex vivo induce SIV-specific immune responses in vivo.

Dendritic cells (DCs) infected with recombinant avipox vectors express the introduced genes and activate antigen-specific T cells. DCs exhibit distinct differentiation-dependent immune functions. Moreover, immature DCs are readily infected by canarypox vectors, but undergo tumor necrosis factor (TNF)-alpha-dependent death, while fewer mature DCs get infected and resist dying. A pilot study was performed using the rhesus macaque system to explore whether immature and mature DCs infected with SIV-recombinant canarypox (vCP180) ex vivo could induce primary virus-specific immune responses in vivo. After subcutaneous (sc) reinjection, functional monocyte-derived DCs migrated to lymph nodes (LNs) within 1-2 days and primed T cells in vivo. This was observed by monitoring dye-labeled DCs in the draining LNs and tetanus toxoid (TT)-specific T cell responses after injection of TT-loaded DCs. DCs from simian immunodeficiency virus (SIV)-naïve rhesus macaques were infected with vCP180 (SIVmac142 gag, pol, and env genes), and sc reinjected into donor animals. Low-level SIV-specific T cell proliferation, but little if any interferon (IFN)-gamma production was detected. DCs pulsed with vCP180 in combination with TT and keyhole limpet hemocyanin (KLH) (to activate additional T cells and provide "helper" cytokines) induced SIV-, TT-, and KLH-specific T cell responses, including IFN-gamma responses not seen when vCP180-carrying DCs were used alone. Interleukin (IL)-10 and low-level antibody responses were also observed. This pilot study provides the proof of principle that sc injected ex vivo SIV-recombinant canarypox-infected DCs safely induce low-level SIV-specific immune responses in vivo.

Human immunodeficiency virus type 1 neutralizing antibody serotyping using serum pools and an infectivity reduction assay.

Classification of human immunodeficiency virus type 1 (HIV-1) by neutralization serotype may be important for the design of active and passive immunization strategies. Neutralizing antibody serotyping is hindered by the lack of standard reagents and assay format, and by the weak activity of many individual sera. To facilitate cross-clade neutralization analysis, we used an infectivity reduction assay (IRA) and selected clade-specific serum (or plasma) pools from subjects infected with clade B and E HIV-1, respectively. Several serum pools were utilized; some were selected for strong neutralizing activity against intraclade viruses and others were derived from conveniently available samples. Against a panel of 51 clade B and E viruses, serum pools displayed strong neutralization of most intraclade viruses and significantly diminished cross-clade neutralization. Results were confirmed against a blinded panel of 20 viruses. The data indicate that the phylogenetic classification of virus subtypes B and E corresponds to two distinct neutralization serotypes. This approach to neutralizing antibody serotyping may be useful in defining the antigenic relationship among viruses from other clades.

Selective increases in HIV-specific neutralizing antibody and partial reconstitution of cellular immune responses during prolonged, successful drug therapy of HIV infection.

Because the immune response to HIV depends on viral gene expression, we examined the HIV-specific immune responses in persons whose viral load after highly active antiretroviral therapy (HAART) was <400 on at least 3 occasions over a 12-month interval. Eleven patients were identified. While there was little change in mean HIV-binding antibody (Ab) titers in this group, two persons mounted increases in HIV envelope-specific binding antibody. Neutralizing antibody (NAb) titers against a panel of HIV-1 primary isolates (BZ167, US1, and CM237) increased post-HAART (80% neutralization titer against US1, p = 0.06; against CM237, p = 0.04). The two persons with large increases in binding antibody also had increases in primary isolate NAb. Roughly half of HAART recipients had significant increases in neutralizing antibody to the primary isolates US1 and CM237. Compared with CD4-matched, non-HAART controls, there were significant increases in NAb against the subtype B primary isolate US1 (p < 0.0009); no increases were seen against more easily neutralized primary isolate BZ167. There were no differences after HAART in antibody-directed cellular cytotoxicity (ADCC). HAART resulted in a partial restoration of lymphoproliferative responses to recall antigens (tetanus and diphtheria). New responses developed to HIV Gag p24. No patient responded to HIV Env gp160 or gp120 either before or after HAART. The data underscore the lack of functional reconstitution of HIV-specific, CD4-mediated responses despite durable suppression of viral replication. In the setting of stable anti-HIV Ab levels, the development of increased NAb in certain individuals suggests that control of the virus by HAART may assist in immune control of HIV.

Analysis of antibody-antigen interactions using surface plasmon resonance.

Accurate analyses of antibody specificities and affinities are critical to understanding their role in antibody-mediated neutralization of HIV-1 in vitro and their potential activity against HIV-1 in vivo. Multiple immunologic tools currently exist for measuring antibody-antigen interactions to include enzyme immunoassays (EIA), Western blotting, radioimmunoassays, and others. However, these techniques all require some form of protein labeling or secondary antibodies for detection of specific binding interactions. Disadvantages of protein labeling involve potential alterations in protein tertiary structure, while the use of secondary detection antibodies prohibits the measurement of binding interactions in real-time. Recently, instruments utilizing the physical principal of surface plasmon resonance (SPR) (BIAcore™, Pharmacia Biosensor, Piscataway, NJ) (1-4) have been developed which allow real-time measurements of protein binding interactions without the use of internal labels or secondary antibodies. Proteins of interest are covalently coupled to a flexible biosensor matrix, and kinetic rate constants can be directly extracted from analyses of association and dissociation binding curves. The relative concentration of immobilized protein can be determined allowing, for example, the comparison of binding efficiency of one antibody against a number of different proteins.

Analysis of Antibody Interactions with HIV-1 Envelope Expressed on the Surface of Acutely Infected H9 Cells.

Protective antibody responses against HIV-1 have yet to be identified or determined. HIV-1 envelope gpl20/gp41 is known to exist as a multimer (tetramers or trimers) on the surface of the virion (1-4). A number of immunoassays have been developed to evaluate HIV-1-specific binding antibody responses using peptides, fusion proteins, and recombinant proteins. Attempts to correlate antibody binding parameters with in vitro HIV-1 neutralization capacity have identified correlations between the presence of V3 antibodies and the capacity to neutralize T-cell line adapted strains of HIV-1. However, no correlation with neutralization of primary HIV-1 isolates and v3 antibodies have been identified (5). Furthermore, binding to monomeric forms of gpl20 has shown little correlation with neutralization of the homologous primary HIV-1 indicating that epitope accessibility and tertiary structure of monomeric forms of gpl20 differs from quaternary structure of membrane expressed oligomeric gpl20/gp41 (6-8).

Collection and Processing of Mucosal Secretions from HIV-1 Infected Women.

The major route of transmission of the human immunodeficiency virus type 1 (HIV-1) worldwide, like other agents of sexually transmitted disease, is via mucosal surfaces of the genital tract through sexual exposures (1-4). It has been hypothesized that immune responses at these sites may be important determinants of protection against HIV-1 (1,2,5). Because of the potential relationship of mucosal immune responses to protection against HIV-1 infection, an assessment of immune responses at these sites in vaccine trial participants will be critical. Proper collection and processing of specimens from mucosal sites is of vital importance to the measurement of mucosal immune responses. This chapter details the collection and processing procedures of specimens from three distinct mucosal compartments in female subjects. Collection techniques for additional mucosal compartments (e.g. semen, rectal, etc.) can be found in ref. 6 (see Note 1).

Collection and processing of mucosal secretions from mice.

The major route of transmission of the human immunodeficiency virus type 1 (HIV-1) worldwide, like other agents of sexually transmitted disease, is via mucosal surfaces of the genital tract through sexual exposures (1-4). It has been hypothesized that immune responses at these sites may be important determinants of protection against HIV-1 (1,2,5). Development of HIV-1 vaccines which elicit local mucosal immune responses may be critical for an effective preventive vaccine. Specific mucosal immune responses elicited after different routes of mucosal immunization in rodents have been examined previously (6-16). Nasal and oral immunization routes have elicited strong mucosal responses to HIV-1 peptides and whole proteins in mice (7,11,17,18,24). Induction of mucosal and systemic immunity to SIV and HIV-1 antigens in primates has also been obtained using a variety of mucosal routes of vaccine administration (19-22).

Measurement of HIV-1 Specific and Total Antibody Secreting Cells by ELISPOT.

B-cells play an important role in protection against pathogens, and they secrete specific antibodies in serum and mucosal secretions upon antigenic stimulation contributing to immune exclusion and clearance of pathogens. The frequency of antibody forming cells (AFC) in specific organs is often a reflection of the route of antigen exposure, i.e., systemic, oral, or intranasal, as well as of antigenic load. Enumeration of AFC was originally performed by plaque-forming cell assay measuring lysis of sheep red blood cells. The nature of this assay, that requires coupling of the antigen to sheep red blood cells (SRBC), made detection of various antigen-specific AFC rather cumbersome. However, the development of the enzyme linked immunodetection of AFCs (ELISPOT) combined with the development of standardized lymphocyte isolation techniques enables detection of AFC secreting antibodies specific for many different antigens and derived from various immunologic effector sites (1-6).