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Empirical testing of candidate vaccines has led to the successful development of a number of lifesaving vaccines. The advent of new tools to manipulate antigens and new methods and vectors for vaccine delivery has led to a veritable explosion of potential vaccine designs. As a result, selection of candidate vaccines suitable for large-scale efficacy testing has become more challenging. This is especially true for diseases such as dengue, HIV, and tuberculosis where there is no validated animal model or correlate of immune protection. Establishing guidelines for the selection of vaccine candidates for advanced testing has become a necessity. A number of factors could be considered in making these decisions, including, for example, safety in animal and human studies, immune profile, protection in animal studies, production processes with product quality and stability, availability of resources, and estimated cost of goods. The “immune space template” proposed here provides a standardized approach by which the quality, level, and durability of immune responses elicited in early human trials by a candidate vaccine can be described. The immune response profile will demonstrate if and how the candidate is unique relative to other candidates, especially those that have preceded it into efficacy testing and, thus, what new information concerning potential immune correlates could be learned from an efficacy trial. A thorough characterization of immune responses should also provide insight into a developer's rationale for the vaccine's proposed mechanism of action. HIV vaccine researchers plan to include this general approach in up-selecting candidates for the next large efficacy trial. This “immune space” approach may also be applicable to other vaccine development endeavors where correlates of vaccine-induced immune protection remain unknown.

Classic preventive vaccines are designed to mimic the effects of natural exposure to microbes. They provide a high level of long-lasting protection against infection in the vast majority of recipients and serve as freestanding preventive measures. Although a classic preventive vaccine remains the ultimate goal of efforts to develop a vaccine for protection against the human immunodeficiency virus (HIV), the enormous genetic diversity and other unique features of the HIV envelope protein have thus far thwarted attempts to identify an effective candidate. However, we have learned from studies of HIV pathogenesis in humans and from animal models that a vaccine that induces strong T-cell–mediated immune responses in the absence of broadly neutralizing antibodies may prove beneficial even if infection is not completely prevented. Vaccine-induced T-cell responses may blunt initial viremia and prevent the early and massive destruction of memory CD4+ T cells that help control infection and prolong disease-free survival. Furthermore, secondary transmission may also be reduced if the vaccine helps to control viral replication; efficiency of transmission is directly related to plasma virus levels. T-cell vaccines represent uncharted territory, and their use may have outcomes that challenge researchers and regulators alike. If proven successful, a disease-modifying HIV vaccine would also present new challenges for the entire public health community, since it would not be a stand-alone preventive measure, as are most classic preventive vaccines. Instead, it would need to be delivered in the context of a comprehensive HIV-prevention program. OB S T AC L E S T O VAC C I NE DE V E L OPM E N T The development of more than two dozen antiretroviral therapies to combat HIV infection has resulted in a dramatic decrease in morbidity and mortality associated with the acquired immunodeficiency syndrome (AIDS) in developed countries and, increasingly, in low- and middle-income countries as these therapies become more widely available. Despite ongoing prevention efforts, however, HIV continues to spread unabated in many parts of the world, with an estimated 14,000 new infections occurring daily. A safe and effective HIV vaccine would be an enormously valuable tool in the campaign to stop the spread of HIV. Most viruses against which successful vaccines have been developed undergo some level of initial replication and dispersal from the portal of entry before the virus reaches its target organ and triggers pathogenic sequelae. During this period, the virus remains vulnerable to eradication by the immune system. When prior immunization or exposure to a virus has elicited virus-specific immunologic memory, the increased speed and intensity of the immune response can prevent or mitigate disease. The nature of the interaction between HIV and the immune system is complex, and the relevance of different immune responses to the control of infection is only partially understood (Fig. 1). The primary stage of HIV infection begins with a burst

This report summarizes the discussions and recommendations from a consultation held in New York City, USA (31 January-2 February 2006) organized by the joint World Health Organization-United Nations Programme on HIV/AIDS HIV Vaccine Initiative and the International AIDS Vaccine Initiative. The consultation discussed issues related to the design and implementation of phase IIB 'test of concept' trials (phase IIB-TOC), also referred to as 'proof of concept' trials, in evaluating candidate HIV vaccines and their implications for future approval and licensure. The results of a single phase IIB-TOC trial would not be expected to provide sufficient evidence of safety or efficacy required for licensure. In many instances, phase IIB-TOC trials may be undertaken relatively early in development, before manufacturing processes and capacity are developed sufficiently to distribute the vaccine on a large scale. However, experts at this meeting considered the pressure that could arise, particularly in regions hardest hit by AIDS, if a phase IIB-TOC trial showed high levels of efficacy. The group largely agreed that full-scale phase III trials would still be necessary to demonstrate that the vaccine candidate was safe and effective, but emphasized that governments and organizations conducting trials should consider these issues in advance. The recommendations from this meeting should be helpful for all organizations involved in HIV vaccine trials, in particular for the national regulatory authorities in assessing the utility of phase IIB-TOC trials in the overall HIV vaccine research and development process. (c) 2007 Lippincott Williams & Wilkins.

Development of a safe and preventive HIV-1 vaccine is a high priority. Recent advances in HIV vaccine development include an improved understanding of HIV envelope structure, development of techniques that enable a detailed analysis of vaccine-induced immune responses in humans, expansion of the pipeline of promising candidate vaccines, and completion of the first vaccine efficacy trials. A common feature of several preventive vaccine strategies in early clinical trials is their ability to attenuate clinical disease rather than completely prevent HIV infection in nonhuman primates. One or more candidate vaccines will likely advance into efficacy trials within the next few years, while efforts to identify new designs that induce broadly neutralizing antibodies continue with incremental success.

A new collaborative model of research is needed to increase resources, to prioritize the RD (ii) to increase the pace, reduce the overlap, and more systematically explore the elements of and delivery systems for vaccines; (iii) to use common standards for the prompt comparative testing of vaccine candidates; (iv) to expand resources for manufacturing vaccine candidates to speed their use in human trials; and (v) to increase the capacity for international clinical trials and to focus this effort toward quickly measuring the effectiveness of vaccine protection as prototype vaccine candidates are identified.

Recent advances in HIV vaccine development include initiation of the first efficacy trials and substantial expansion of the preclinical pipeline. Several preclinical candidate vaccines have induced strong cellular immune responses and provided impressive protection against AIDS in non-human primate models; however, candidates that induce broadly neutralizing antibodies remain elusive.

An HIV vaccine has thus far been elusive. Drs. Margaret Johnston and Anthony Fauci remain cautiously optimistic that a substantial increase in our understanding of HIV infection and disease will lead to creative ideas about how to design an effective HIV vaccine. Dr. Anthony Fauci discusses the 30-year search for an HIV vaccine, recent setbacks, and prospects for future success. Dr. Fauci is the director of the National Institute of Allergy and Infectious Diseases, Bethesda, MD.

The need to broaden research directed at answering fundamental questions in HIV vaccine discovery through laboratory, nonhuman primate (NHP), and clinical research has recently been emphasized. In addition, the importance of attracting and retaining young researchers, developing better NHP models, and more closely linking NHP and clinical research is being stressed. In an era of a level budget for biomedical research at the U.S. National Institutes of Health (NIH), HIV/AIDS vaccine research efforts will need to be carefully prioritized such that resources to energize HIV vaccine discovery can be identified. This article summarizes progress and challenges in HIV vaccine research, the priorities arising from a recent summit at NIAID, and the actions needed, some already under way, to address those priorities.

Because of a unique combination of challenges facing human immunodeficiency virus vaccine developers, a number of traditional and novel vaccine designs are being evaluated essentially in parallel. Monomeric proteins, poxvirus vectors, peptides, and particle-based candidate vaccines have entered or will soon enter human trials. Other designs are at earlier stages of development. All candidates evaluated to date in phase I/II human trials have proven safe and immunogenic. One or more of the most promising designs will soon progress to 'test of concept' clinical trials to determine efficacy.

Since our natural immune response to HIV infection is ineffective, a key goal for an HIV vaccine is to induce a response different from that induced by natural infection — an “unnatural immunity,” involving production of broadly neutralizing antibodies.

Developing a vaccine against the human immunodeficiency virus (HIV) poses an exceptional challenge. There are no documented cases of immune-mediated clearance of HIV from an infected individual, and no known correlates of immune protection. Although non-human primate models of lentivirus infection have provided valuable data about HIV pathogenesis, such models do not predict HIV vaccine efficacy in humans. The combined lack of a predictive animal model and undefined biomarkers of immune protection against HIV necessitate that vaccines to this pathogen be tested directly in clinical trials. Adaptive clinical trial designs can accelerate vaccine development by rapidly screening out poor vaccines while extending the evaluation of efficacious ones, thereby improving the characterization of promising vaccine candidates and the identification of correlates of immune protection.

Since the discovery of human immunodeficiency virus (HIV) in 1983, significant progress has been made toward the discovery, development, and licensing of anti-HIV drugs. In vitro screens against whole virus are now being complemented by screens against specific viral targets, resulting in the development of clinical candidates acting at several critical stages of the viral life cycle. Despite these advances, clinical therapy remains largely palliative. In addition, it has recently been recognized that HIV resistance to most drugs may pose even greater obstacles. Moreover, emerging data on immunopathogenesis raise the possibility that even if virus was eliminated from an infected individual, the patient's immune system might not be capable of restoration to normal function. In the face of such obstacles, deeper insights into the pathogenic mechanisms of disease, aggressive exploitation of those mechanisms for therapeutic gain, and continued commitment of both public and private sectors to support and collaborate in this research are needed.

Analysis of the structure-anti-HIV activity correlations of halo dideoxynucleosides (ddN's) in the public domain was accomplished through computer substructure searching, retrieval and sorting of in vitro anti-HIV data. In the survey, selectivity index (ratio of cytotoxicity to the potency in inhibiting HIV replication in vitro) was used to rank compounds in congeneric groups. Factors contributing to the anti-HIV activity, e.g. the nature and location of the halogen on the sugar or the base and its stereochemical configuration, could not be generalized for all the halogenated ddN's. Conclusions were drawn for specific classes within the pyrimidine and purine series, with compounds further divided into halo substitutions at the sugar 2',3',4'-positions or in the pyrimidine or purine ring systems. At the 3'-position, only a fluoro substitution enhanced the activity of the dideoxypyrimidine nucleosides. A 2'-ara fluoro substituent increased the activity of purine ddN's but decreased activity of pyrimidine ddN's. Halogenation of the side chain in acyclic adenine and 2,6-diaminopurine nucleosides improved their anti-HIV activity. Halo substitution at the 6- or 2'-ara position of selected purine ddN's resulted in compounds with increased lipophilicity, chemical stability and retention of anti-HIV activity. The number of halodideoxynucleosides tested as anti-HIV reagents suggested that this class of compounds is well studied.

Since the identification of HIV in 1984, the search for a safe and effective vaccine has been relentless. While investigator-initiated research has provided substantial information regarding HIV disease and pathogenesis, and over two dozen drugs are licensed in the USA to treat HIV, the global epidemic continues unabated. Early in HIV vaccine research, the pharmaceutical industry took the initiative to produce products for clinical testing. As the likelihood of a quick success decreased, private investment waned. The public sector responded with novel mechanisms to engage industry while continuing to support academic investigators. HIV vaccine research continues to rely on the creativity of individual investigators, as well as collaborations that vary in size and complexity and offer opportunities for the efficient use of resources and accelerated progress.

The discovery of an HIV-1 vaccine is a high priority. Recent advances in HIV vaccine development include an improved understanding about virus biology and structure, and the development of quantitative techniques that enable a detailed analysis of vaccine-induced immune responses in humans. The preclinical vaccine pipeline looks healthy, and a common feature of the new vaccine strategies is their ability to attenuate clinical disease rather than prevent HIV infection in nonhuman primates. Human clinical trials to evaluate the safety and immunogenicity of these vaccine candidates and strategies are being actively pursued.

Although animal models have been useful in guiding vaccine development, HIV/AIDS models have not yielded a clear correlate of immunity nor given consistent results on the potential efficacy of various vaccine approaches. Further development and improved uniformity in the use of animal models would maximize their potential to meet the urgent worldwide need for a safe and effective HIV vaccine.

Several lines of evidence now argue strongly that a successful HIV vaccine does not need to block infection completely. It has to mimic what already occurs in a minority of unvaccinated individuals--namely, rapid clearance of infection or reduction of viral load to a level that does not produce symptoms or permit transmission to others. The various vaccine candidates are reviewed.

The optimal use of biological response modifiers (BRMs) in human immunodeficiency virus (HIV)-related disease depends on knowledge of the molecular basis of the immune deficiencies and dysregulations that occur during the course of the infection; evidence for the role of viral products and cytokines in the suppression of immune function is discussed. Immunebased therapies are currently being explored alone and in combination with drugs targeted to HIV and associated opportunistic infections and malignancies. These therapies include hematopoietic growth factors for the management of drug toxicities, cytokines, antigen- and cell-based therapies, and synthetic immunomodulators. The entry of additional BRMs into clinical trials for HIV-disease can be facilitated by well-designed preclinical studies that address special problems related to the disease. including the need for concomitant therapy for the spectrum of disease manifestations encountered.

In this Policy Forum, the [authors][1] respond to [ Burton et al .][2] by asserting that the ongoing efficacy trial of ALVAC-HIV (vCP1521) and AIDSVAX B/E in Thailand is scientifically justified, morally correct, and strategically important. No laboratory assay or animal model has been validated as a predictor of the efficacy of HIV vaccines in humans, thus efficacy trials are essential to test bona fide vaccine hypotheses. Although there is a real chance that the prime-boost combination may not be efficacious, there is a very high probability that information gained will significantly advance HIV vaccine development. Established scientific principles and appropriate processes fully support the decision to proceed with this efficacy trial. [1]: http://www.sciencemag.org/cgi/content/full/303/5660/961 [2]: http://www.sciencemag.org/cgi/content/short/303/5656/316

Antisera elicited by immunization of rabbits with 4'-aminomethyl-trioxsalen (AMT)-modified poly(A,U) complexed with methylated bovine serum albumin was characterized in competition radioimmunoassays (RIA) and enzyme-linked immunosorbent assays (ELISA). AMT-poly(A,U) was over 10,000-fold more reactive than unmodified poly(A,U) or AMT alone. The antiserum cross-reacted to varying extents with AMT-modified-RNA's and -DNA's. The presence of AMT-uridine usually assured strong reactivity. The amino group of AMT contributed to antibody binding to a small degree. Binding was not significantly affected by high ionic strength, suggesting that binding does not involve ion pair formation. Murine encephalomyocarditis virus replicative intermediates, as well as cellular RNA and DNA were modified by psoralen in intact cells, suggesting that EMCV RNA and cellular RNA's in intact cells possess detectable stretches of base pairs. The antibodies described here will be useful in studying the secondary and tertiary structure of RNA's in vitro and in intact cells.

5'-O-Monophosphoryladenylyl(2' goes to 5')adenylyl-(2' goes to 5')adenosine [(2' goes to 5')(pA)3] antagonizes the protein synthesis inhibitory effects of 5'-O-triphosphoryladenylyl-(2' goes to 5')adenylyl(2' goes to 5')adenosine by preventing activation of the (2' goes to 5')oligo(a)-activated ribonuclease which degrades mRNA. The oligoribonucleotide (2' goes to 5')(pA)3 also antagonizes the translational inhibitory capacity of poly(I).poly(C) in extracts of interferon-treated L cells, suggesting that (2' goes to 5')oligo(A) is the primary mediator of the protein synthesis inhibitory effects of poly(I).poly(C) in this cell-free system.

A competition immunoenzymometric assay for 2',5'-oligoadenylate was developed and employed to measure the interferon-inducible enzyme 2',5'-oligoadenylate synthetase in cell extracts. Microtiter plates coated with a novel conjugate of p5'A2'p5'A2'p5'A and N-(2-aminoethyl)-carbamylmethylated-Ficoll (AECM-Ficoll) bound rabbit polyclonal or mouse monoclonal antibody directed against 2',5'-oligoadenylate. Binding was inhibited by soluble 2',5'-oligoadenylate. Estimates of 2',5'-oligoadenylate concentrations based on inhibition of antibody binding compared favorably with those obtained using a protein synthesis inhibition assay. Low concentrations of 2',5'-oligoadenylate synthesized in vitro by extracts of human peripheral mononuclear cells were conveniently estimated using less than or equal to 10(6) cells. Virtually identical results were obtained when either total extract or synthetase bound to poly(I) . poly(C)-agarose was used for the in vitro incubation. When peripheral mononuclear cells were incubated in vitro with interferon, the normally low levels of 2',5'-oligo(A) synthetase rose dramatically. The assay was employed to measure synthetase levels in peripheral mononuclear cells isolated from patients with systemic lupus erythematosus. Some of these patients were found to have elevated levels of 2',5'-oligoadenylate synthetase.

2',5'-Oligoadenylate synthetase was induced 3-2000-fold in spleen, liver, kidney and brain of NIH Swiss mice injected intravenously with 2-200 micrograms of the misaligned dsRNA, poly(I).poly(C12,U). Levels of 2',5'-oligoadenylates extracted from these tissues were also elevated, although the amount of 2',5'-oligoadenylates extracted did not correlate directly with the amount of enzyme present. These results suggest that double-stranded portions of the misaligned polymer survived intracellularly and activated the 2',5'-oligoadenylate synthetase, and that the level of dsRNA may contribute to the control of 2',5'-oligoadenylate metabolism.

Raman spectra of model compounds and of 2',5'-oligoadenylates in D2O were utilized to assign the Raman bands of 2',5'-oligoadenylates. The Raman spectra of A2'pA2'pA, pA2'pA2'pA, and pppA2'pA2'pA contained features that were similar to those of adenosine, adenosine 5'-monophosphate (AMP), and adenosine 5'-triphosphate, respectively. When AMP and pA2'pA2'pA were titrated from pH 2 to 9, the normalized Raman intensity of their ionized (980 cm-1) and protonated (1080 cm-1) phosphate bands revealed similar pKa's for the 5'-monophosphates. The Raman spectrum of pA2'pA2'pA was altered slightly by elevations in temperature, but not in a manner supporting the postulate that 2-5A possesses intermolecular base stacking. Major differences in the Raman spectrum of 2',5'- and 3',5'-oligoadenylates were observed in the 600-1200-cm-1 portion of the spectrum that arises predominately from ribose and phosphate vibrational modes. Phosphodiester backbone modes in A3'pA3'pA and pA3'pA3'pA produced a broad band at 802 cm-1 with a shoulder at 820 cm-1, whereas all 2',5'-oligoadenylates contained a major phosphodiester band at 823 cm-1 with a shoulder at 802 cm-1. The backbone mode of pppA2'pA2'pA contained the sharpest band at 823 cm-1, suggesting that the phosphodiester backbone may be more restrained in the biologically active, 5'-triphosphorylated molecule. The Raman band assignments for 2',5'-oligoadenylates provide a foundation for using Raman spectroscopy to explore the mechanism of binding of 2',5'-oligoadenylates to proteins.

A rapid and convenient assay for adenylyl(2′ → 5′)adenosine(A2′p5′A) or adenylyl(3′ → 5′)adenosine(A3′p5′A) phosphodiesterase activities is described. The dinucleotides A3′p5′A and A2′p5′A were labeled to a high specific activity by means of a catalytic-exchange procedure. Degradation studies of each of these labeled dinucleotides showed an asymmetrical distribution of label between the two adenine bases. Enzymatic degradation of [ 3 H]A3′p5′A or [ 3 H]A2′p5′A could be quantitated by first digesting the reaction products with bacterial alkaline phosphatase and then adding a slurry of DEAE-Sephadex. Under conditions described, adenosine did not adsorb to the resin, whereas dinucleotides as well as AMP did adsorb. As a consequence, when liquid scintillation fluid was added to the DEAE-Sephadex reaction mixture slurry, the radioactivity of the dinucleotides and AMP was severely quenched. This permitted a direct estimation of the amount of adenosine liberated during the phosphodiesterase degradation and subsequent alkaline phosphatase digestion. This method was applied to the measurement of A2′p5′A degrading activities in extracts of mouse L cells. Extracts from control mouse L cells were as active in degrading A2′p5′A as extracts from interferon pretreated cells.

A number of oligonucleotides have been synthesized and evaluated as antagonists of the protein synthesis inhibitory effects of 2-5A in order to gain information on the oligonucleotide structural features involved in binding to the 2-5A-activated endoribonuclease. The base moieties, the ribose-phosphate backbone and the 5′-monophosphate group of p5′A2′p5′A2′p5′A are strategic regions for binding to the endonuclease; however, rather extensive modification of the 2′-terminal ribose unit is possible without adversely affecting interaction with the endonuclease. Analogues of p5′A2′p5′A2′p5′A, p5′A2′p5′ A2′p5′A2′p5′A or ppp5′A2′p5′A2′p5′A2′p5′A, in which the 2′-terminal ribose has been modified to an N-hexylmorpholine ring have significantly enhanced activity as antagonists of 2-5A action or as inhibitors of protein synthesis. Such analogues are much more resistant to degradation by the phosphodiesterase activity found in L cell extracts. These analogues of p5′A2′p5′A2′p5′A are also effective antagonists of the inhibition of protein synthesis caused by dsRNA in extracts of interferon-treated L cells, and their use demonstrates that 2-5A is the primary mediator of inhibition caused by dsRNA. In accord with this finding, the synthetic dsRNA, poly(A). poly(dUfl), an activator of the protein P 1 kinase but not the 2-5A synthetase, was a potent inhibitor of protein synthesis in rabbit reticulocyte lysates, but was without discernable activity in extracts of interferon-treated L cells. Thus, phosphorylation of eIF-2α and/or protein does not appear to be a sufficient condition for protein synthesis inhibition in extracts of interferon-treated L cells.

Publisher Summary This chapter presents the methodology used to extract IF mRNA, partially purify IF mRNA, and assay IF mRNA by translation in Xenopus laevis oocytes. It is shown that total cytoplasmic RNA from Newcastle disease virus (NDV)-induced L-929 cells can program the synthesis of interferon in Xenopus laevis oocytes. Moreover, if this RNA is further purified by sucrose-formamide gradient centrifugation, a linear dose-response relationship is obtained between the amount of RNA injected into the oocyte and the amount of interferon translated. A similar quantitative relationship has also been established for the oocyte and wheat germ systems programmed with human interferon mRNA. These observations have facilitated a study of the mechanism of superinduction of human fibroblast interferon production. For studying the mechanism of interferon priming on interferon production in NDV-induced L-929 cells, a advantage of this simple quantitative assay to study the kinetics and intracellular distribution of interferon mRNA (IF mRNA) is presented. Priming by interferon did not affect IF mRNA transcription or stability, but rather its rate qf translation in NDV-induced mouse L cells.

Publisher Summary This chapter presents an assay for 2',5'-oligoadenylate synthetase that provides reproducibly high yields of (2'-5')-oligo(A) when crude cell extracts are employed, and provides an opportunity for variation of nucleic acid activator. This method is based on the binding of (2'-5')-oligo(A) synthetase from crude extracts to 2',5'-ADP-Sepharose at 4°. After binding, the resin is washed to remove unbound molecules, and the resin bound synthetase is incubated at 30° in the presence of ATP and a nucleic acid activator. The (2'-5')-oligo(A) in the reaction supernatant is then assayed by its ability to inhibit cell free protein synthesis. This method is a convenient and quantitative alternative to the assay of (2'-5')-oligo(A) synthetase from crude extracts on poly(I) . poly(C)-Sepharose. In extracts that contain high levels of inactivating enzymes, both methods may be superior to a solution assay in that both resin assays afford a high degree of purification in the binding step.

The enzyme oligo(2′–5′)adenylate synthetase, when activated by double-stranded RNA, polymerizes ATP into the novel oligonucleotide (2′–5′)ppp(Ap)nA. We describe conditions for assay of this enzyme in crude extracts of a human lymphoblastoid cell line, Namalwa. The production of (2′–5′)ppp(Ap)nA by Namalwa extracts was 3–5 times greater than the production by extracts of interferon pretreated mouse L cells, and 700 fold higher than the production by extracts of untreated mouse L cells. The relatively high level of oligo(2′–5′)adenylate synthetase in Namalwa cells was not attributable solely to their constitutive secretion of low levels of interferon. Analysis of the size distribution of the oligomers formed at different times suggested that the enzyme can add ATP to a free pppApA. Infection by Newcastle disease virus or treatment with interferon raised the apparent synthetase levels only marginally. Experiments that employed antibody to interferon suggested that the interferon must be externalized from the NDV-infected cell to induce maximal synthetase levels.

To define the epitopes involved in binding anti-oligonucleotide antibodies, several hybridomas producing monoclonal antibodies directed against 2',5'-oligoadenylate were established. A solid-phase enzyme-linked immunoassay that employed microtiter wells coated with Ficoll-2',5'-oligoadenylate conjugates proved useful in screening and characterizing hybridoma supernatants. Control experiments demonstrated that the conjugates were irreversibly adsorbed to polystyrene wells under the conditions employed in the assay. Reactivity of monoclonal antibodies with numerous analogues of 2',5'-oligoadenylate was measured by using a competition assay. Several monoclonal antibodies originating from different mice immunized with the same or different immunogens possessed distinctive fine specificities. At least one 2',5'-phosphodiester bond was important in forming each epitope, suggesting that the ribose phosphate backbone is a critical element in defining an antigenic domain of an oligonucleotide. The purine bases were also important, and modification of the bases had varied effects on the extent of antibody recognition. The length of the oligonucleotide and the nature of the termini were also of some importance. In several instances the modification created by linkage of 2',5'-oligoadenylate to carrier protein also contributed to the determinant. The monoclonal antibody most specific for 2',5'-oligoadenylates was relatively insensitive to ionic strength. In contrast, a monoclonal antibody with a 2',5'-oligopurine specificity appeared to bind 2',5'-oligoadenylate through one ion pair, whereas the binding of a monoclonal antibody with a low degree of base specificity appeared to bind through two ion pairs. The results demonstrated that 2',5'-linked oligoadenylate-protein complexes possess at least three distinct oligonucleotide-related antigenic surfaces that can be recognized with high apparent affinity by monoclonal antibodies. A model for the three epitopes is presented.