Active immunization with tau epitope in a mouse model of tauopathy induced strong antibody response together with improvement in short memory and pSer396-tau pathology.

Abnormal tau hyperphosphorylation and its aggregation into neurofibrillary tangles are a hallmark of tauopathies, neurodegenerative disorders that include Alzheimer's disease (AD). Active and passive Tau-immunotherapy has been proposed as a therapeutic approach to AD with mixed results. One of the limitations of active immunotherapy may be associated with the mediocre immunogenicity of vaccines that are not inducing therapeutically potent titers of antibodies. The aim of this study was to test the efficacy of an anti-tau vaccine, AV-1980R/A composed of N terminal peptide of this molecule fused with an immunogenic MultiTEP platform and formulated in a strong adjuvant, AdvaxCpG in a Tg4510 mouse model of tauopathy. Experimental mice were immunized with AV-1980R/A and a control group of mice were injected with adjuvant only. Nontransgenic and tetracycline transactivator (tTA) transgenic littermates were included as baseline controls to contrast with the tau phenotype. Active immunization with AV-1980R/A induced very strong anti-tau humoral immune responses in both nontransgenic and transgenic mice with evidence of IgG in brains of AV-1980R/A vaccinated mice. These experimental animals displayed an improvement in short-term memory during a novel object recognition test. However, impairments in other behavioral tasks were not prevented by AV-1980R/A vaccinations. At the same time, high titers of anti-tau antibodies reduced hyperphosphorylated pSer396 tau but did not lower the level of other phosphorylated tau species in the brains of AV-1980R/A vaccinated mice. These data indicate that active immunotherapy with an N-terminal Tau epitope was only partially effective in improving cognition and reducing pathology in the stringent Tg4510 mouse model of tauopathy.

Protection of chimpanzees from high-dose heterologous HIV-1 challenge by DNA vaccination.

Novel approaches for the generation of more effective vaccines for HIV-1 are of significant importance. In this report we analyze the immunogenicity and efficacy of an HIV-1 DNA vaccine encoding env, rev and gag/pol in a chimpanzee model system. The immunized animals developed specific cellular and humoral immune responses. Animals were challenged with a heterologous chimpanzee titered stock of HIV-1 SF2 virus and followed for 48 weeks after challenge. Polymerase chain reaction coupled with reverse transcription (RT-PCR) results indicated infection in the control animal, whereas those animals vaccinated with the DNA constructs were protected from the establishment of infection. These studies serve as an important benchmark for the use of DNA vaccine technology for the production of protective immune responses.

DNA prime-protein boost increased the titer, avidity and persistence of anti-Abeta antibodies in wild-type mice.

Recently, we reported that a DNA vaccine, composed of three copies of a self B cell epitope of amyloid-beta (Abeta(42)) and the foreign T-cell epitope, Pan DR epitope (PADRE), generated strong anti-Abeta immune responses in wild-type and amyloid precursor protein transgenic animals. Although DNA vaccines have several advantages over peptide-protein vaccines, they induce lower immune responses in large animals and humans compared with those in mice. The focus of this study was to further enhance anti-Abeta(11) immune responses by developing an improved DNA vaccination protocol of the prime-boost regimen, in which the priming step would use DNA and the boosting step would use recombinant protein. Accordingly, we generated DNA and recombinant protein-based epitope vaccines and showed that priming with DNA followed by boosting with a homologous recombinant protein vaccine significantly increases the anti-Abeta antibody responses and do not change the immunoglobulin G1 (IgG1) profile of humoral immune responses. Furthermore, the antibodies generated by this prime-boost regimen were long-lasting and possessed a higher avidity for binding with an Abeta(42) peptide. Thus, we showed that a heterologous prime-boost regimen could be an effective protocol for developing a potent Alzheimer's disease (AD) vaccine.

DNA, but not protein vaccine based on mutated BORIS antigen significantly inhibits tumor growth and prolongs the survival of mice.

The ideal immunological target for cancer vaccine development would meet the criteria of tumor specificity, immunogenicity and vital dependency of the tumor on the functional activities of the antigenic target so as to avoid antigenic loss by mutation. Given that at face value the brother of regulator of imprinted sites (BORIS) transcription factor meets these criteria, we have developed a mutant variant of this molecule (mBORIS) that lacks tumorigenic ability, while retaining immunogenic epitopes that elicits responses against histologically irrelevant tumor cells. Here we compared vaccine strategies employing as an immunogen either mBORIS recombinant protein formulated in a strong Th1-type adjuvant, QuilA or DNA encoding this immunogen along with plasmids expressing interleukin (IL)12/IL18 molecular adjuvants. In both groups of vaccinated mice induction of tumor-specific immunity (antibody response, T-cell proliferation, cytokine production, T-cell cytotoxicity) as well as ability to inhibit growth of the aggressive breast cancer cell line and to prolong survival of vaccinated animals have been tested. We determined that DNA, but not recombinant protein vaccine, induced potent Th1-like T-cell recall responses that significantly inhibited tumor growth and prolongs the survival of vaccinated mice. These studies demonstrate that DNA immunization is superior to recombinant protein strategy and provide a clear guidance for clinical development of a cancer vaccine targeting what appears to be a universal tumor antigen.

Intracellular adhesion molecule-1 modulates beta-chemokines and directly costimulates T cells in vivo.

The potential roles of adhesion molecules in the expansion of T cell-mediated immune responses in the periphery were examined using DNA immunogen constructs as model antigens. We coimmunized cDNA expression cassettes encoding the adhesion molecules intracellular adhesion molecule-1 (ICAM-1), lymphocyte function associated-3 (LFA-3), and vascular cell adhesion molecule-1 (VCAM-1) along with DNA immunogens, and we analyzed the resulting antigen-specific immune responses. We observed that antigen-specific T-cell responses can be enhanced by the coexpression of DNA immunogen and adhesion molecules ICAM-1 and LFA-3. Coexpression of ICAM-1 or LFA-3 molecules along with DNA immunogens resulted in a significant enhancement of T-helper cell proliferative responses. In addition, coimmunization with pCICAM-1 (and more moderately with pCLFA-3) resulted in a dramatic enhancement of CD8-restricted cytotoxic T-lymphocyte responses. Although VCAM-1 and ICAM-1 are similar in size, VCAM-1 coimmunization did not have any measurable effect on cell-mediated responses. These results suggest that ICAM-1 and LFA-3 provide direct T-cell costimulation. These observations are further supported by the finding that coinjection with ICAM-1 dramatically enhanced the level of interferon-gamma (IFN-gamma) and beta-chemokines macrophage inflammatory protein-1alpha (MIP-1alpha), MIP-1beta, and regulated on activation normal T-cell expression and secreted (RANTES) produced by stimulated T cells. Through comparative studies, we observed that ICAM-1/LFA-1 T-cell costimulatory pathways are independent of CD86/CD28 pathways and that they may synergistically expand T-cell responses in vivo.

CD8 positive T cells influence antigen-specific immune responses through the expression of chemokines.

The potential roles of CD8(+) T-cell-induced chemokines in the expansion of immune responses were examined using DNA immunogen constructs as model antigens. We coimmunized cDNA expression cassettes encoding the alpha-chemokines IL-8 and SDF-1alpha and the beta-chemokines MIP-1alpha, RANTES, and MCP-1 along with DNA immunogens and analyzed the resulting antigen-specific immune responses. In a manner more similar to the traditional immune modulatory role of CD4(+) T cells via the expression of Th1 or Th2 cytokines, CD8(+) T cells appeared to play an important role in immune expansion and effector function by producing chemokines. For instance, IL-8 was a strong inducer of CD4(+) T cells, indicated by strong T helper proliferative responses as well as an enhancement of antibody responses. MIP-1alpha had a dramatic effect on antibody responses and modulated the shift of immune responses to a Th2-type response. RANTES coimmunization enhanced the levels of antigen-specific Th1 and cytotoxic T lymphocyte (CTL) responses. Among the chemokines examined, MCP-1 was the most potent activator of CD8(+) CTL activity. The enhanced CTL results are supported by the increased expression of Th1 cytokines IFN-gamma and TNF-alpha and the reduction of IgG1/IgG2a ratio. Our results support that CD8(+) T cells may expand both humoral and cellular responses in vivo through the elaboration of specific chemokines at the peripheral site of infection during the effector stage of the immune response.

Peptide mimicry of carbohydrate epitopes on human immunodeficiency virus.

Cancer-related, mucin-type carbohydrate epitopes, principally mannose and sialo-syl residues, are expressed on the envelope protein gp 160 of the human immunodeficiency virus (HIV). Anticarbohydrate antibodies directed toward these and other carbohydrate epitopes are known to neutralize HIV-1 infection by cell-free virus. Carbohydrates, however, being T cell-independent antigens, typically elicit diminished immune responses. To overcome this potential draw back, we have examined the ability of peptides that mimic such epitopes to elicit immune responses that cross-react with carbohydrate structures. We report that mouse polyclonal antisera generated against peptides that mimic mucin-related carbohydrate epitopes have anti-HIV-1 activity. Generation of antibodies was not lr-gene restricted, as at least two different strains of mice. Balb/c (H-2d) and C57Bl/6 (H-2b), responded equally to the peptides. The antipeptide sera displayed neutralizing activity against HIV-I/MN and HIV-I/3B viral strains. This neutralization was as good as human anti-HIV sera. These results indicate that peptide mimics of carbohydrates provide a novel strategy for the further development of reagents that elicit immune responses to carbohydrate epitopes associated with many infectious organisms and tumor cells.

Engineering of in vivo immune responses to DNA immunization via codelivery of costimulatory molecule genes.

Nucleic acid immunization is a novel vaccination technique to induce antigen-specific immune responses. We have developed expression cassettes for cell surface markers CD80 and CD86, two functionally related costimulatory molecules that play an important role in the induction of T cell-mediated immune responses. Coimmunization of these expression plasmids, along with plasmid DNA encoding for HIV-1 antigens, did not result in any significant change in the humoral response; however, we observed a dramatic increase in cytotoxic T-lymphocyte (CTL) induction as well as T-helper cell proliferation after the coadministration of CD86 genes. In contrast, coimmunization with a CD80 expression cassette resulted in a minor, but positive increase in T-helper cell or CTL responses. This strategy may be of value for the generation of rationally designed vaccines and immune therapeutics.

Molecular and immunological analysis of genetic prostate specific antigen (PSA) vaccine.

Nucleic acid immunization has been investigated as immunotherapy for infectious diseases as well as for treating specific types of cancers. In this approach, nucleic acid expression cassettes are directly inoculated into the host, whose transfected cells become the production source of novel and possibly immunologically foreign protein. We have developed a DNA vaccine construct which encodes for PSA by cloning a cDNA for PSA into a mammalian expression vector under control of a CMV promoter. We investigated and characterized the immunogenicity of PSA DNA expression cassettes in mice. PSA-specific immune responses induced in vivo by immunization were characterized by enzyme-linked immunosorbent assay (ELISA), T helper proliferation cytotoxic T lymphocyte (CTL), and flow cytometry assays. We observed a strong and persistent antibody response against PSA for at least 180 days following immunization. In addition, a significant T helper cell proliferation was observed against PSA protein. Using synthetic peptides spanning the PSA open frame, we identified four dominant T helper epitopes of PSA. Furthermore, immunization with PSA plasmid induced MHC Class I CD8+ T cell-restricted cytotoxic T lymphocyte response against tumor cell targets expressing PSA. The prostate represents a very specific functional organ critical for reproduction but not for the health and survival of the individual. Understanding the immunogenicity of PSA DNA immunization cassettes offers insight into the possible use of this tumor-associated antigen as a target for immunotherapy. These results demonstrate the ability of the genetic PSA to serve as a specific immune target capable of generating both humoral and cellular immune responses in vivo.

Antigenic and immunological mimicry of peptide mimotopes of Lewis carbohydrate antigens.

Peptides may substitute for carbohydrates in reactions with carbohydrate-specific molecules. Recently, we found that peptides containing aromatic residues mimic mucin and histo-blood group related carbohydrate epitopes, eliciting polyclonal responses cross-reactive with bacterial and viral antigens that express these carbohydrate forms. These results demonstrate that peptides can function in in vivo and in vitro models as carbohydrate surrogate antigens. To further explore the nature of the antigenic and immunogenic properties of such mimotopes, synthetic peptides with aromatic amino acids were tested to delineate reactivity patterns with several anti-neolactoseries monoclonal antibodies (MAbs). These MAbs recognize biologically important conformations of the histo-blood group related Lewis antigens expressed on the surface of a variety of human cancers. Results by ELISA demonstrate that the MAbs can distinguish particular peptide motifs that include the sequences GGIYYPYDIYYPYDIYYPYD, GGIYWRYDIYWRYDIYWRYD and GGIYYRYDIYYRYDIYYRYD. Substitution of Arg by Pro diminished the reactivity of the anti-Lewis Y (LeY) MAb BR55-2. Binding of LeY to BR55-2 was inhibitable by the Arg containing peptides. Serum against all three peptides displayed reactivity with synthetic histo-blood group related antigen probes. Immunologic presentation of the peptides as multiple antigen peptides (MAPs) improved peptide ability to induce LeY specific immune responses. Serum bound to human tumor cells that preferentially expressed neolactoseries antigens, but not to normal tissues. Immunoprecipitation of human breast tumor cell lysates before and after treatment with tunicamycin confirmed serum carbohydrate binding. The anti-peptide sera mediated tumor cell killing by complement mediated cytotoxicity. These results indicate that mapping peptide epitopes with anti-carbohydrate antibodies can lend to defining antibody fine specificities that can go undetected by screening of carbohydrate antigens alone. In addition, these results confirm that peptides and carbohydrates can bind to the same antibody binding site and that peptides can structurally mimic salient features of carbohydrate epitopes.

Macrophage colony-stimulating factor can modulate immune responses and attract dendritic cells in vivo.

Studies have indicated that professional APCs in the periphery, such as dendritic cells and macrophages, play an important role in initiating DNA vaccine-specific immune responses. To engineer the immune response induced by DNA vaccines in vivo we investigated the modulatory effects of codelivering growth factor genes for the hematopoietic APCs along with DNA vaccines. Specifically, we examined the effects on the antigen-specific immune responses following the codelivery of the gene expression cassettes for M-CSF, G-CSF, and GM-CSF along with HIV-1 DNA immunogen constructs. We observed that coimmunization with GM-CSF increased the antibody response and resulted in a significant enhancement of lymphoproliferative response. Furthermore, among all coinjection combinations, we found that M-CSF coinjections resulted in a high level of CTL enhancement. This enhancement of CTL responses observed from the coinjection with M-CSF was CD8+ T cell dependent and was associated with the presence of CD11c+ cells at the site of injection and with the antigen-specific induction of the beta-chemokine MIP-1beta, suggesting a role for this chemokine in CTL induction. These results suggest that hematopoietic growth factors should be further studied as potential adjuvants for in vivo modulators of immune responses.

Cytokine molecular adjuvants modulate immune responses induced by DNA vaccine constructs for HIV-1 and SIV.

DNA or nucleic acid immunization has been shown to induce both antigen-specific cellular and humoral immune responses in vivo. Moreover, immune responses induced by DNA immunization can be enhanced and modulated by the use of molecular adjuvants. To further engineer the immune response in vivo, we investigated the induction and regulation of immune responses from the codelivery of Thl cytokines (interleukin-2 [IL-2] and IL-12), Th2 cytokines (IL-4 and IL-10), and granulocyte-macrophage colony-stimulating factor (GM-CSF) genes along with a DNA vaccine construct encoding for simian immunodeficiency virus (SIV) gag/pol proteins. We observed that coinjection with IL-2, IL-4, IL-10, and GM-CSF resulted in increased levels of antigen-specific antibodies. In addition, we found that coinjection with cytokine genes drove the immune responses toward a more Thl or Th2 phenotype. We also observed that coadministration of IL-2, IL-12, and GM-CSF genes resulted in a dramatic enhancement of Th proliferation responses. Moreover, coimmunization with IL-12 genes resulted in a dramatic enhancement of antigen-specific cytotoxic T lymphocyte (CTL) responses. These results support the potential utility of molecular adjuvants in DNA vaccine regimens.

DNA inoculation as a novel vaccination method against human retroviruses with rheumatic disease associations.

There are a number of rheumatologic manifestations of human retroviral infections associated with human immunodeficiency virus type I (HIV-I) and the human T-cell leukemia virus type I (HTLV-I) including arthritis, Sjøgren's syndrome-like symptoms as well as other varied autoimmune phenomena. Infection with HTLV-1 may be directly involved in the etiology and/or pathogenesis of an arthritic condition similar to rheumatoid arthritis. We have been characterizing a new vaccination strategy against human retroviral infections, designated DNA inoculation. This procedure involves the intramuscular injection of DNA plasmids which express specific human retroviral antigens. This technique results in the development of humoral and cellular immune responses against these proteins. Specifically, this method has been successfully used to develop immune responses against HIV-I and HTLV-I. The availability of rat and rabbit infection models for HTLV-I, coupled with the successful development of immune responses in these animals after DNA inoculation with an HTLV-I envelope expressing plasmid, will allow the efficacy of this vaccination technique to be evaluated with protection against in vivo viral challenge as an endpoint.

Molecular cloning, expression, and biological characterization of an HTLV-II envelope glycoprotein: HIV-1 expression is permissive for HTLV-II-induced cell fusion.

The human T cell leukemia virus II (HTLV-II) is a type C retrovirus closely related to the human transforming retrovirus HTLV-I. In contrast to HTLV-I, the role of HTLV-II in human disease is controversial. However, HTLV-II infection has been documented in several cases of a clinically benign hairy cell leukemia and has also been suggested as a cofactor for HIV-1 disease progression. We report that an HTLV-II isolate (designated FLW) derived from a serum-positive white male can induce cell fusion and significant cytopathic effects in tissue culture. This HTLV-II isolate induced syncytium formation with human T and B cell lines, several human fibroblast cell lines, and, interestingly, HIV-1- and HIV-2-infected cell lines. To elucidate the role in the FLW envelope in this phenomenon, we have cloned the envelope glycoproteins gp46 and gp21 of this isolate. The envelope glycoproteins expressed in the absence of the rest of the viral genome were sufficient to drive syncytium formation in vitro, and preserved the cellular tropism for syncytium formation observed with the native retroviral isolate. Amino acid (aa) sequence analysis demonstrated 88% overall similarity with other HTLV-II envelope glycoproteins. Interestingly, only cells infected by HIV-I, but not parental H9 cells, form syncytia with FLW env-transfected cells as well as with HTLV-II/FLW-infected BJAB-WH cells. Furthermore, antibodies directed at the CD4 receptor failed to inhibit the induction of giant cell formation, implying that the FLW envelope protein was responsible for driving syncytium formation in this system. These observations may be important for the understanding of the processes involved in human retroviral-mediated syncytium formation and may suggest a mechanism whereby HTLV-II could influence the disease process in individuals dually infected with HIV-1 and HTLV-II.

An HIV type 2 DNA vaccine induces cross-reactive immune responses against HIV type 2 and SIV.

We have previously reported on the generation of specific functional immune responses after inoculation of animals with expression vectors encoding HIV-1 genes. This article provides the details of the first application of this new technology to induce immune responses against HIV-2. This virus is molecularly and serologically distinct from HIV-1 and is in fact more closely related to the simian immunodeficiency virus (SIV). Anti-HIV-2 and SIV antibodies were induced in mice of three different haplotypes following a single intramuscular inoculation with an HIV-2/ROD envelope glycoprotein expression vector (pcEnv-2). Boosting of animals with pcEnv-2 induced both anti-HIV-2 neutralizing antibodies and T cell-proliferative responses against HIV-2 and SIVmac proteins. We compared the humoral and cellular immune responses of mice injected with pcEnv-2 and then boosted with either the homologous DNA construct or a recombinant Env protein. Animals boosted with pcEnv-2 generated B and T cell immune responses as strong as those of mice boosted with recombinant gp140 protein in adjuvant. Finally, cellular immune responses were significantly increased with the coadministration of pcEnv-2 and a plasmid expressing interleukin 12. We therefore conclude that DNA plasmid inoculation induces cross-reactive anti-HIV-2 and anti-SIVmac immune responses in mice. This technology should be further investigated as a potential vaccine component for this human pathogen.

In vitro and in vivo tumor growth suppression by HIV-1 Vpr.

We have previously reported that the human immunodeficiency virus type 1 (HIV-1) regulatory gene vpr induces differentiation of rhabdomyosarcoma (embryonal muscle tumor cell line) cells, an effect that is accompanied by reduced proliferative capacity of the transfected cells. In this report, we examine the effect of Vpr expression on several different tumor cell lines derived from unique lineages. These tumor cells display different patterns of modulated oncogenes including both ras and p53 mutations. Here we demonstrate that the growth of tumor cells in vitro and in vivo is arrested by the expression of HIV-1 Vpr. Expression of Vpr in several human tumor cell lines upon transfection resulted in an accumulation of cells in the G2/M phase of cell cycle with altered cellular morphology, including an increase in adherence, and growth arrest, consistent with a differentiated phenotype. Vpr expression in B78/H1 cells results in a marked reduction in colony formation in vitro and an associated reduction in melanin synthesis by the cells. Vpr-transfected melanoma cells inoculated into syngenic C57BL/6 mice showed a markedly reduced ability to form tumors in vivo. These results suggest that this retroviral regulatory gene has broad tumor suppressor effects, likely mediated by transcriptional regulation of the state of the host cell.

Murine monoclonal antibodies biologically active against the amino region of HIV-1 gp120: isolation and characterization.

The human immunodeficiency virus (HIV)-1 envelope glycoprotein is synthesized as a precursor (gp160) and subsequently cleaved to generate the external gp120 and transmembrane gp41 glycoproteins. Both gp120 and gp41 have been demonstrated to mediate critical functions of HIV, including viral attachment and fusion with the cell membrane. The antigenic variability of the HIV-1 envelope glycoprotein has presented a significant problem in the design of appropriate and successful vaccines and offers one explanation for the ability of HIV to evade immune surveillance. Therefore, the development and characterization of functional antibodies against conserved regions of the envelope glycoprotein is needed. Because of this need, we generated a panel of murine monoclonal antibodies (MuMabs) against the HIV-1 envelope glycoprotein. To accomplish this, we immunized Balb/C mice with a recombinant glycoprotein 160 (gp160) that was synthesized in a baculovirus expression system. From the growth-positive hybridomas, three MuMabs were generated that demonstrated significant reactivity with recombinant gp120 but failed to show reactivity against HIV-1 gp41, as determined by enzyme-linked immunosorbent assay (ELISA). Using vaccinia constructs that synthesize variant truncated subunits of gp160, we were able to map reactivity of all three of the Mabs (ID6, AC4, and AD3) to the first 204 residues of gp120 (i.e., the N terminus of gp120) via Western blot analysis. Elucidation of the epitopes for these Mabs may have important implications for inhibition of infection by HIV-1. Our initial attempts to map these Mabs with linear epitopes have not elucidated a specific antigenic determinant; however, several physical characteristics have been determined that suggest a continuous surface epitope. Although these antibodies failed to neutralize cell-free or cell-associated infection by HIV-1, they did mediate significant antibody-dependent cellular cytotoxicity (ADCC) activity, indicating potential therapeutic utility. In summary, these data suggest the identification of a potentially novel site in the first 200 aa of gp120 that mediates ADCC.

Peptide mimicry of adenocarcinoma-associated carbohydrate antigens.

Carbohydrate antigens have been identified as significant antigens in many human tumors either by analyzing antibodies in patients' sera or by using monoclonal antibodies of either mouse or human origin. Three carbohydrate epitopes present on cancer-associated mucins [sialyl-Lewis A (SLA), sialyl-Lewis X (SLX), and sialyl-Tn (STn)] may have functional significance in metastasis. Subsequently, these antigens are considered as targets for active specific immunotherapy. Carbohydrates, as T-cell-independent antigens, often elicit diminished immune responses. To overcome this drawback, carbohydrates are typically coupled to protein carriers to elicit immunoglobulin G (IgG) responses as opposed to low-affinity IgM responses, which often times accompanies carbohydrate-based immunizations. In addition, some complex carbohydrates are difficult to synthesize. This latter aspect is further magnified if one considers that clustering of epitopes on neoglycoproteins must be emulated in the synthesis process, leading to multiple presentation or tandem repeats of the synthetic carbohydrate immunogen. Here, we examine the hypothesis that peptides that mimic carbohydrates might be developed to induce immune responses that target and mediate the killing of tumor cells, particularly breast cancer cells in an adjuvant-type setting. We have found that carbohydrate-mimicking peptides retain carbohydrate-like conformations, inducing anti-carbohydrate immune responses against breast tumor cells and mediating their killing by a complement-dependent mechanism.

Monoclonal antibodies define a cellular antigen involved in HTLV-I infection.

The exact mechanism by which the human T cell leukemia viruses (HTLV) infects target cells remains unclear; although some molecules have been identified to be important in viral infection and entry. To investigate these phenomena, we generated a panel of monoclonal antibodies (MAb) against a B cell line (BJAB-WH) which is highly permissive for infection with HTLV. These MAb have been used to further characterize the membrane molecules important for HTLV infection. Three of these MAb designated 4.2.3, 3.3.10, and 11.2.3 were capable of inhibiting syncytium formation induced in human B and T cell lines (i.e., BJAB-WH and SupT-1, respectively) by co-culture with HTLV-I infected MT-2 cells. All of these MAbs immunoprecipitated a 80-85 kDa antigen from the lysates of metabolically labeled BJAB-WH but not from BJAB-CC/84, a noninfectible target cell. The binding of these MAb with different HTLV target cells was analyzed and compared with binding of polyclonal monospecific antisera to the same cell lines. A 80-85 kDa membrane glycoprotein was isolated with an immunoaffinity chromatographic column constructed with MAbs 4.2.3 and 3.3.10. This cellular antigen was capable of inhibiting HTLV I/MT-2 induced fusion. This is the first direct demonstration that a 80-85 kDa cellular glycoprotein is directly involved in HTLV I/II infection and syncytium formation.