Determinants of CD4 cell count decline after treatment interruption

Patients who had started HAART (Highly Active Anti-Retroviral Treatment) under previous aggressive DHHS guidelines (1997) underwent a life-long continuous HAART that was associated with many short term as well as long term complications. Many interventions attempted to reduce those complications including intermittent treatment also called pulse therapy. Many studies were done to study the determinants of rate of fall in CD4 count after interruption as this data would help guide treatment interruptions. The data set used here was a part of a cohort study taking place at the Johns Hopkins AIDS service since January 1984, in which the data were collected both prospectively and retrospectively. The patients in this data set consisted of 47 patients receiving via pulse therapy with the aim of reducing the long-term complications. ^ The aim of this project was to study the impact of virologic and immunologic factors on the rate of CD4 loss after treatment interruption. The exposure variables under investigation included CD4 cell count and viral load at treatment initiation. The rates of change of CD4 cell count after treatment interruption was estimated from observed data using advanced longitudinal data analysis methods (i.e., linear mixed model). Using random effects accounted for repeated measures of CD4 per person after treatment interruption. The regression coefficient estimates from the model was then used to produce subject specific rates of CD4 change accounting for group trends in change. The exposure variables of interest were age, race, and gender, CD4 cell counts and HIV RNA levels at HAART initiation. ^ The rate of fall of CD4 count did not depend on CD4 cell count or viral load at initiation of treatment. Thus these factors may not be used to determine who can have a chance of successful treatment interruption. CD4 and viral load were again studied by t-tests and ANOVA test after grouping based on medians and quartiles to see any difference in means of rate of CD4 fall after interruption. There was no significant difference between the groups suggesting that there was no association between rate of fall of CD4 after treatment interruption and above mentioned exposure variables. ^

Boosting with recombinant vaccinia increases immunogenicity and protective efficacy of malaria DNA vaccine

To enhance the efficacy of DNA malaria vaccines, we evaluated the effect on protection of immunizing with various combinations of DNA, recombinant vaccinia virus, and a synthetic peptide. Immunization of BALB/c mice with a plasmid expressing Plasmodium yoelii (Py) circumsporozoite protein (CSP) induces H-2Kd-restricted CD8+ cytotoxic T lymphocyte (CTL) responses and CD8+ T cell- and interferon (IFN)-��-dependent protection of mice against challenge with Py sporozoites. Immunization with a multiple antigenic peptide, including the only reported H-2Kd-restricted CD8+ T cell epitope on the PyCSP (PyCSP CTL multiple antigenic peptide) and immunization with recombinant vaccinia expressing the PyCSP induced CTL but only modest to minimal protection. Mice were immunized with PyCSP DNA, PyCSP CTL multiple antigenic peptide, or recombinant vaccinia expressing PyCSP, were boosted 9 wk later with the same immunogen or one of the others, and were challenged. Only mice immunized with DNA and boosted with vaccinia PyCSP (D-V) (11/16: 69%) or DNA (D-D) (7/16: 44%) had greater protection (P < 0.0007) than controls. D-V mice had significantly higher individual levels of antibodies and class I-restricted CTL activity than did D-D mice; IFN-�� production by ELIspot also was higher in D-V than in D-D mice. In a second experiment, three different groups of D-V mice each had higher levels of protection than did D-D mice, and IFN-�� production was significantly greater in D-V than in D-D mice. The observation that priming with PyCSP DNA and boosting with vaccinia-PyCSP is more immunogenic and protective than immunizing with PyCSP DNA alone supports consideration of a similar sequential immunization approach in humans.

Rational engineering of a miniprotein that reproduces the core of the CD4 site interacting with HIV-1 envelope glycoprotein

Protein���protein interacting surfaces are usually large and intricate, making the rational design of small mimetics of these interfaces a daunting problem. On the basis of a structural similarity between the CDR2-like loop of CD4 and the ��-hairpin region of a short scorpion toxin, scyllatoxin, we transferred the side chains of nine residues of CD4, central in the binding to HIV-1 envelope glycoprotein (gp120), to a structurally homologous region of the scorpion toxin scaffold. In competition experiments, the resulting 27-amino acid miniprotein inhibited binding of CD4 to gp120 with a 40 ��M IC50. Structural analysis by NMR showed that both the backbone of the chimeric ��-hairpin and the introduced side chains adopted conformations similar to those of the parent CD4. Systematic single mutations suggested that most CD4 residues from the CDR2-like loop were reproduced in the miniprotein, including the critical Phe-43. The structural and functional analysis performed suggested five additional mutations that, once incorporated in the miniprotein, increased its affinity for gp120 by 100-fold to an IC50 of 0.1���1.0 ��M, depending on viral strains. The resulting mini-CD4 inhibited infection of CD4+ cells by different virus isolates. Thus, core regions of large protein���protein interfaces can be reproduced in miniprotein scaffolds, offering possibilities for the development of inhibitors of protein���protein interactions that may represent useful tools in biology and in drug discovery.

Superoxide-mediated clastogenesis and anticlastogenic effects of exogenous superoxide���dismutase

Superoxide-mediated clastogenesis is characteristic for various chronic inflammatory diseases with autoimmune reactions and probably plays a role in radiation-induced clastogenesis and in the congenital breakage syndromes. It is consistently prevented by exogenous superoxide dismutase (SOD), but not by heat-inactivated SOD, indicating that the anticlastogenic effect is related to the catalytic function of the enzyme. Increased superoxide production by activated monocytes/macrophages is followed by release of more long-lived metabolites, so-called clastogenic factors, which contain lipid peroxidation products, unusual nucleotides of inosine, and cytokines such as tumor necrosis factor ��. Since these components are not only clastogenic, but can stimulate further superoxide production by monocytes and neutrophils, the genotoxic effects are self-sustaining. It is shown here that anticlastogenic effects of exogenous SOD are preserved despite extensive washing of the cells and removal of all extracellular SOD. Using flow cytometry and confocal laser microscopy, rapid adherence of the fluorescently labeled enzyme to the cell surface could be observed with slow uptake into the cell during the following hours. The degree of labeling was concentration and time dependent. It was most important for monocytes, compared with lymphocytes, neutrophils, and fibroblasts. The cytochrome c assay showed significantly diminished O2��� production by monocytes, pretreated with SOD and washed thereafter. The preferential and rapid binding of SOD to monocytes may be of importance not only for the superoxide-mediated genotoxic effects, described above, but also from a therapeutic standpoint. It can explain the observation that beneficial effects of injected SOD lasted for weeks and months despite rapid clearance of the enzyme from the blood stream according to pharmacodynamic studies.

Measuring the diaspora for virus-specific CD8+ T cells

The CD8+ T cell diaspora has been analyzed after secondary challenge with an influenza A virus that replicates only in the respiratory tract. Numbers of DbNP366- and DbPA224-specific CD8+ T cells were measured by tetramer staining at the end of the recall response, then followed sequentially in the lung, lymph nodes, spleen, blood, and other organs. The extent of clonal expansion did not reflect the sizes of the preexisting memory T cell pools. Although the high-frequency CD8+ tetramer+ populations in the pneumonic lung and mediastinal lymph nodes fell rapidly from peak values, the ���whole mouse��� virus-specific CD8+ T cell counts decreased only 2-fold over the 4 weeks after infection, then subsided at a fairly steady rate to reach a plateau at about 2 months. The largest numbers were found throughout in the spleen, then the bone marrow. The CD8+DbNP366+ and CD8+DbPA224+ sets remained significantly enlarged for at least 4 months, declining at equivalent rates while retaining the nucleoprotein > acid polymerase immunodominance hierarchy characteristic of the earlier antigen-driven phase. Lowest levels of the CD69 ���activation marker��� were detected consistently on virus-specific CD8+ T cells in the blood, then the spleen. Those in the bone marrow and liver were intermediate, and CD69hi T cells were very prominent in the regional lymph nodes and the nasal-associated lymphoid tissue. Any population of ���resting��� CD8+ memory T cells is thus phenotypically heterogeneous, widely dispersed, and subject to broad homeostatic and local environmental effects irrespective of epitope specificity or magnitude.

Effects of inefficient cleavage of the signal sequence of HIV-1 gp 120 on its association with calnexin, folding, and intracellular transport.

The HIV-1 envelope glycoprotein gp120 displays inefficient intracellular transport, which is caused by its retention in the endoplasmic reticulum. Coexpression in insect cells (Sf9) of HIV-1 gp120 with calnexin has shown that their interaction was modulated by the signal sequence of HIV-1 gp120. gp120, with its natural signal sequence, showed a prolonged association with calnexin with a t1/2 of greater than 20 min. Replacement of the natural signal sequence with the signal sequence from mellitin led to a decreased time of association of gp120 with calnexin (t1/2 < 10 min). These different times of calnexin association coincided both with the folding of gp120 as measured by the ability of bind CD4 and with endoplasmic reticulum to Golgi transport as analyzed by the acquisition of partial endoglycosidase H resistance. Using a monospecific antibody to the HIV-1 gp120 natural signal peptide, we showed that calnexin associated with N-glycosylated but uncleaved gp120. Only after dissociation from calnexin was gp120 cleaved, but very inefficiently. Only the small proportion of signal-cleaved gp120 molecules acquired transport competence and were secreted. This is the first report demonstrating the effect of the signal sequence on calnexin association.

Phenotypic and physiologic characterization of transgenic mice expressing interleukin 4 in the lung: lymphocytic and eosinophilic inflammation without airway hyperreactivity.

To investigate the contribution of interleukin-4 (IL-4) to airway inflammation in vivo and to explore directly its relationship to airway reactivity, we created transgenic mice in which the murine cDNA for IL-4 was regulated by the rat Clara cell 10 protein promoter. Expression was detected only in the lung and not in thymus, heart, liver, spleen, kidney, or uterus. The expression of IL-4 elicited hypertrophy of epithelial cells of the trachea, bronchi, and bronchioles. Hypertrophy is due, at least in part, to the accumulation of mucus glycoprotein. Histologic examination of parenchyma revealed multinucleated macrophages and occasional islands of cells consisting largely of eosinophils or lymphocytes. Analysis of lung lavage fluid revealed the presence of a leukocytic infiltrate consisting of lymphocytes, neutrophils and eosinophils. Mice expressing IL-4 had greater baseline airway resistance but did not demonstrate hyperreactivity to methacholine. Thus, the expression of IL-4 selectively within the lung elicits an inflammatory response characterized by epithelial cell hypertrophy, and the accumulation of macrophages, lymphocytes, eosinophils, and neutrophils without resulting in an alteration in airway reactivity to inhaled methacholine.

Unimpaired autoreactive T-cell traffic within the central nervous system during tumor necrosis factor receptor-mediated inhibition of experimental autoimmune encephalomyelitis.

The critical role of tumor necrosis factor (TNF) as a mediator in autoimmune inflammatory processes is evident from in vivo studies with TNF-blocking agents. However, the mechanisms by which TNF, and possibly also its homologue lymphotoxin alpha, contributes to development of pathology in rheumatoid arthritis and Crohn disease and in animal models like experimental autoimmune encephalomyelitis is unclear. Possibilities include regulation of vascular adhesion molecules enabling leukocyte movement into tissues or direct cytokine-mediated effector functions such as mediation of tissue damage. Here we show that administration of a TNF receptor (55 kDa)-IgG fusion protein prevented clinical signs of actively induced experimental autoimmune encephalomyelitis. Significantly, the total number of CD4+ T lymphocytes isolated from the central nervous system of clinically healthy treated versus diseased control animals was comparable. By using a CD45 congenic model of passively transferred experimental autoimmune encephalomyelitis to enable tracking of myelin basic protein-specific effector T lymphocytes, prevention of clinical signs of disease was again demonstrated in treated animals but without quantitative or qualitative impediment to the movement of autoreactive T lymphocytes to and within the central nervous system. Thus, despite the uninterrupted movement of specific T lymphocytes into the target tissue, subsequent disease development was blocked. This provides compelling evidence for a direct effector role of TNF/lymphotoxin alpha in autoimmune tissue damage.

Investiga����o do papel de SIGIRR/IL-1R8 no crosstalk entre c��lulas tumorais e o infiltrado leucocit��rio

C��lulas tumorais desenvolvem diversas estrat��gias para escapar da identifica����o e elimina����o pelo sistema imune. Dessa forma, a investiga����o dos mecanismos envolvidos na comunica����o celular no microambiente tumoral e na desregula����o local do sistema imune �� cr��tica para uma melhor compreens��o da progress��o da doen��a e para o desenvolvimento de alternativas terap��uticas mais eficazes. N��s aqui demonstramos que SIGIRR/IL-1R8, um importante regulador negativo de receptores de Interleucina-1 (ILRs) e receptores do tipo Toll (TLRs), apresenta express��o aumentada em uma linhagem celular epitelial mam��ria transformada pela superexpress��o do oncogene HER2 e em tumores prim��rios de mama, e promove o crescimento tumoral e met��stase atrav��s da modula����o da inflama����o associada ao c��ncer e da atenua����o da resposta imune antitumoral. Observamos que IL-1R8 tem sua express��o correlacionada com HER2 em tecidos mam��rios e sua alta express��o �� fator de pior progn��stico em c��ncer de mama de baixo grau. Notavelmente, n��veis aumentados de IL-1R8 foram observados especialmente nos subtipos HER2+ e Luminais de tumores de mama, e sua express��o aumentada em c��lulas epiteliais de mama transformadas por HER2 diminui a ativa����o da via de NF-&#954;B e a express��o de diferentes citocinas pro-inflamat��rias (IL-6, IL-8, TNF, CSF2, CSF3 e IFN-&#946;1). Meio condicionado de c��lulas transformadas por HER2, mas n��o de variantes celulares com o gene IL-1R8 silenciado, induz a polariza����o de macr��fagos para o fen��tipo M2 e inibe a ativa����o de c��lulas NK. Em um modelo murino transg��nico de tumorig��nese espont��nea mediada por HER2, MMTV-neu, verificamos que a defici��ncia de IL-1R8 (IL-1R8-/-neu) retardou o aparecimento de tumores e reduziu a incid��ncia, a carga tumoral e a dissemina����o metast��tica. Contudo, n��o foram observadas diferen��as significativas no crescimento tumoral quando animais IL-1R8-/-neu receberam medula ��ssea de animais IL-1R8+/+, confirmando um papel importante da express��o de IL-1R8 em c��lulas n��o hematopoi��ticas na tumorig��nese da mama. Tumores IL-1R8+/+neu apresentaram maiores n��veis de citocinas pr��-inflamat��rias como IL-1&#946; e VEGF, e menores n��veis da citocina imunomodulat��ria IFN-&#947;. Al��m disso, tumores que expressavam IL-1R8 apresentaram menor infiltrado de c��lulas NK maduras, c��lulas dendr��ticas (DCs) e linf��citos T-CD8+ e um maior infiltrado de macr��fagos M2 e linf��citos T-CD4+. Coletivamente, esses resultados indicam que a express��o de IL-1R8 em tumores de mama pode representar um novo mecanismo de escape da resposta imune e suportam IL-1R8 como potencial alvo terap��utico.

An��lise da express��o da prote��na NY-ESO-1 no melanoma cut��neo

INTRODU����O: o c��ncer �� a doen��a que mais mata pessoas com idade abaixo de 85 anos e �� um problema de sa��de p��blica. Os tumores podem expressar em determinada fase de seu desenvolvimento prote��nas an��malas que podem ser alvo de m��todos diagn��sticos e de interven����es terap��uticas. A express��o de NY-ESO-1 �� detectada em 20 a 40% dos melanomas. H�� evid��ncias que esta express��o �� mais freq��ente em tumores de est��gios mais avan��ados e est�� associada a um pior progn��stico. OBJETIVOS: determinar a frequ��ncia de express��o da prote��na NY-ESO-1 no melanoma cut��neo e tentar correlacion��-la com o ��ndice de Breslow, aspectos histopatol��gicos do melanoma, incluindo o infiltrado linfoc��tico tumoral, e a morbi-mortalidade dos pacientes. M��TODOS: o presente estudo �� longitudinal de coorte retrospectiva e foi realizado de agosto de 2009 a outubro de 2015. Foram selecionados 89 melanomas de 87 pacientes do Ambulat��rio de Tumores do Departamento de Dermatologia da FMUSP, divididos em 3 grupos, sendo: grupo 1: 34 melanomas com ��ndice de Breslow <= 1,0 mm; grupo 2: 29 melanomas com ��ndice de Breslow entre 1,1 - 4,0 mm e grupo 3: 26 melanomas com ��ndice de Breslow >= 4,0 mm. As l��minas dos exames an��tomo-patol��gicos destes pacientes foram revisadas quanto ao diagn��stico de melanoma, seu ��ndice de Breslow e a presen��a de infiltrado linfoc��tico tumoral. A seguir, realizou-se exame de imunohistoqu��mica para a determina����o da presen��a do ant��geno NY-ESO-1 em todos os 89 tumores coletados e em mais 20 nevos (11 displ��sicos e 9 intrad��rmicos) escolhidos ao acaso. Atrav��s da revis��o dos dados do prontu��rio, foram obtidos os dados cl��nicos de: idade, sexo, ra��a, fototipo da pele, local de aparecimento do melanoma, status do linfonodo sentinela quando realizado, desenvolvimento de met��stases e sobrevida dos pacientes. Os dados an��tomo-patol��gicos do tumor analisados foram: tipo histol��gico, presen��a de ulcera����o, e tipo de infiltrado linfoc��tico tumoral. Nos melanomas que apresentavam infiltrado linfoc��tico tumoral, foram realizados testes imunohistoqu��micos para pesquisa de c��lulas CD3+, CD8+, FoxP3+ e CD8+FoxP3+ (duplamente positivas). RESULTADOS: O ant��geno NY-ESO-1 esteve presente em 19% dos melanomas cut��neos prim��rios e n��o foi detectado em nenhum dos 20 nevos pesquisados. A express��o do ant��geno NY-ESO-1 esteve estatisticamente relacionada a tumores com espessuras maiores. Apresentou tamb��m uma associa����o inversa com o tipo extensivo superficial em rela����o aos outros tipos histol��gicos. O infiltrado linfoc��tico tumoral dos melanomas NY-ESO-1 positivos continha menor n��mero de c��lulas CD3+, que se encontravam isoladas ou arranjadas em pequenos grupos de at�� 5 c��lulas, o que contrastava significantemente com os tumores NY-ESO-1 negativos, com maior densidade de c��lulas CD3+, dispostas em grandes grupos, com 6 ou mais c��lulas. A express��o da prote��na NY-ESO-1 n��o esteve associada �� idade, ao sexo, ao fototipo, ao s��tio prim��rio do tumor, �� presen��a de ulcera����o, ao status do linfonodo sentinela, ao desenvolvimento de met��stases ou �� sobrevida. CONCLUS��ES: H�� express��o de NY-ESO-1 em uma porcentagem consider��vel dos melanomas, principalmente nos mais espessos. O menor n��mero de c��lulas CD3+ no infiltrado linfoc��tico tumoral, acrescido ao fato destas c��lulas estarem isoladas ou em pequenos grupos, sugere que embora imunog��nico, a express��o do ant��geno NY-ESO-1 n��o resulta num est��mulo eficaz do sistema imune no combate ao tumor. O desenvolvimento de uma vacina para estes pacientes poder��, no futuro, aumentar as possibilidades terap��uticas do melanoma

Role of IFN-gamma in immune responses to Candida albicans infections

Candida albicans is the most frequent etiologic agent that causes opportunistic fungal infections called candidiasis, a disease whose systemic manifestation could prove fatal and whose incidence is increasing as a result of an expanding immunocompromised population. Here we review the role of interferon-gamma (IFN-��) in host protection against invasive candidiasis. This cytokine plays an essential role in both the innate and adaptive arms of the immune response to candidiasis. We focus on recent progress on host-pathogen interactions leading to the production of IFN-�� by host cells. IFN-�� is produced by CD4 Th1, CD8, ���� T, and natural killer (NK) cells, essentially in response to both IL-12 and/or IL-18; more recently, a subset of C. albicans-specific Th17 cells have been described to produce both IL-17 and IFN-��. IFN-�� plays an important role in the regulation of the immune system as well as in the control of the infectious process, as it is required for optimal activation of phagocytes, collaborates in the generation of protective antibody response, and favors the development of a Th1 protective response.

M��canismes d'action des cellules stromales m��senchymateuses dans le traitement de la r��action du greffon contre l'h��te

La maladie du greffon contre l���h��te (GvHD) est un effet secondaire s��rieux de la transplantation de cellules souches h��matopo����tiques (HSCT). Cette maladie entraine une haute mortalit�� et ses sympt��mes sont d��vastateurs. Les traitements actuels de la GvHD comportent plusieurs produits, tels les corticost��ro��des, mais ces derniers sont immunosuppresseurs et leurs effets secondaires sont aussi tr��s dommageables pour les patients et leur gu��rison. Les cellules stromales m��senchymateuses (MSC) repr��sentent une alternative ou une addition potentielle de traitement pour la GvHD et ces cellules ne semblent pas poss��der les effets secondaires des traitements classiques. Un nombre important d�����tudes cliniques faisant l���objet des MSC ont ��t�� enregistr��es. Malgr�� cet engouement, le m��canisme de leur immunomodulation reste encore �� ��lucider. Notre objectif est donc de mieux d��finir ce m��canisme. Nous avons utilis�� un mod��le simplifi�� pour simuler la GvHD in vitro. Ce mod��le se base sur la stimulation de lymphocytes CD4+ par des cellules dendritiques allog��niques. La mesure de la prolif��ration de ces cellules stimul��es sert d���indicateur de leur r��activit��. Selon les r��sultats obtenus par la technologie CRISPR de g��nie g��n��tique, les MSC exerceraient leur immunosuppression sur les cellules T CD4+ principalement par la s��cr��tion de l���enzyme IDO1. Les MSC seraient ��galement capables d���induire certaines cellules CD4+ en cellules r��gulatrices, un processus ind��pendant de la s��cr��tion d���IDO1. Toutefois, ces cellules ne semblent pas correspondre aux cellules Treg conventionnelles.

Differential proliferative response of NKT cell subpopulations to in vitro stimulation in presence of different cytokines

Human Valpha24(+)Vbeta11(+) NKT (NKT) cells have immune regulatory activities associated with rejection of tumors, infections and control of autoimmune diseases. They can be stimulated to proliferate using alpha-galactosylceramide (KRN7000) and have the potential for therapeutic manipulation. Subpopulations of NKT cells (CD4(+)CD8(-), CD4(-)D8(+) and CD4(-)CD8(-)) have functionally distinctive Th1/Th2 cytokine profiles and their relative numbers following stimulation may influence the Th1/Th2 balance, which may result in or prevent disease. We aimed to determine the effect of different cytokines in culture during stimulation of NKT cells on the relative proportions of NKT cell subpopulations. Our results show that all NKT cell subpopulations expanded following stimulation with KRN7000 and IL-2, IL-7, IL-1 2 or IL-15. Expansion capacity differed between subpopulations, resulting in different relative proportions of CD4(+) and CD4(-) NKT cell subpopulations, and this was influenced by the cytokine used for stimulation. A Th1-biased environment was observed after stimulation of NKT cells. NKT cells expanded under all conditions evaluated demonstrated significant cytotoxicity against U937 tumor cells. In view of the potential for NKT cell subsets to alter the balance of Th1 and Th2 environment, these data provide insights into the effects of NKT cell manipulation for possible therapeutic applications in different disease settings.

Phase 1 study of HPV16-specific immunotherapy with E6E7 fusion protein and ISCOMATRIX (TM) adjuvant in women with cervical intraepithelial neoplasia

Purpose: Persistent infection of cervical epithelium with high risk human papillomavirus (HPV) results in cervical intraepithelial neoplasia (CIN) from which squamous cancer of the cervix can arise. A study was undertaken to evaluate the safety and immunogenicity of an HPV 16 immunotherapeutic consisting of a mixture of HPV16 E6E7 fusion protein and ISCOMATRIX(TM) adjuvant (HPV16 Immunotherapeutic) for patients with CIN. Experimental design: Patients with CIN (n = 3 1) were recruited to a randomised blinded placebo controlled dose ranging study of immunotherapy. Results: Immunotherapy was well tolerated. Immunised subjects developed HPV16 E6E7 specific immunity. Antibody, delayed type hypersensitivity, in vitro cytokine release, and CD8 T cell responses to E6 and E7 proteins were each significantly greater in the immunised subjects than in placebo recipients. Loss of HPV16 DNA from the cervix was observed in some vaccine and placebo recipients. Conclusions : The HPV16 Immunotherapeutic comprising HPV16E6E7 fusion protein and ISCOMATRIX(TM) adjuvant is safe and induces vaccine antigen specific cell mediated immunity. (C) 2004 Elsevier Ltd. All rights reserved.

theta-defensins prevent HIV-1 Env-mediated fusion by binding gp41 and blocking 6-helix bundle formation

Retrocyclin-1, a 0-defensin, protects target cells from human immunodeficiency virus, type 1 (HIV-1) by preventing viral entry. To delineate its mechanism, we conducted fusion assays between susceptible target cells and effector cells that expressed HIV-1 Env. Retrocyclin-1 (4 mu M) completely blocked fusion mediated by HIV-1 Envs that used CXCR4 or CCR5 but had little effect on cell fusion mediated by HIV-2 and simian immunodeficiency virus Envs. Retrocyclin-1 inhibited HIV-1 Env-mediated fusion without impairing the lateral mobility of CD4, and it inhibited the fusion of CD4-deficient cells with cells bearing CD4-independent HIV-1 Env. Thus, it could act without cross-linking membrane proteins or inhibiting gp120-CD4 interactions. Retrocyclin-1 acted late in the HIV-1 Env fusion cascade but prior to 6-helix bundle formation. Surface plasmon resonance experiments revealed that retrocyclin bound the ectodomain of gp41 with high affinity in a glycan-independent manner and that it bound selectively to the gp41 C-terminal heptad repeat. Native-PAGE, enzyme-linked immunosorbent assay, and CD spectroscopic analyses all revealed that retrocyclin-1 prevented 6-helix bundle formation. This mode of action, although novel for an innate effector molecule, resembles the mechanism of peptidic entry inhibitors based on portions of the gp41 sequence.