eCD4-Ig Variants That More Potently Neutralize HIV-1.

The human immunodeficiency virus type 1 (HIV-1) entry inhibitor eCD4-Ig is a fusion of CD4-Ig and a coreceptor-mimetic peptide. eCD4-Ig is markedly more potent than CD4-Ig, with neutralization efficiencies approaching those of HIV-1 broadly neutralizing antibodies (bNAbs). However, unlike bNAbs, eCD4-Ig neutralized all HIV-1, HIV-2, and simian immunodeficiency virus (SIV) isolates that it has been tested against, suggesting that it may be useful in clinical settings, where antibody escape is a concern. Here, we characterize three new eCD4-Ig variants, each with a different architecture and each utilizing D1.22, a stabilized form of CD4 domain 1. These variants were 10- to 20-fold more potent than our original eCD4-Ig variant, with a construct bearing four D1.22 domains (eD1.22-HL-Ig) exhibiting the greatest potency. However, this variant mediated less efficient antibody-dependent cell-mediated cytotoxicity (ADCC) activity than eCD4-Ig itself or several other eCD4-Ig variants, including the smallest variant (eD1.22-Ig). A variant with the same architecture as the original eCD4-Ig (eD1.22-D2-Ig) showed modestly higher thermal stability and best prevented the promotion of infection of CCR5-positive, CD4-negative cells. All three variants, and eCD4-Ig itself, mediated more efficient shedding of the HIV-1 envelope glycoprotein gp120 than did CD4-Ig. Finally, we show that only three D1.22 mutations contributed to the potency of eD1.22-D2-Ig and that introduction of these changes into eCD4-Ig resulted in a variant 9-fold more potent than eCD4-Ig and 2-fold more potent than eD1.22-D2-Ig. These studies will assist in developing eCD4-Ig variants with properties optimized for prophylaxis, therapy, and cure applications.IMPORTANCE HIV-1 bNAbs have properties different from those of antiretroviral compounds. Specifically, antibodies can enlist immune effector cells to eliminate infected cells, whereas antiretroviral compounds simply interfere with various steps in the viral life cycle. Unfortunately, HIV-1 is adept at evading antibody recognition, limiting the utility of antibodies as a treatment for HIV-1 infection or as part of an effort to eradicate latently infected cells. eCD4-Ig is an antibody-like entry inhibitor that closely mimics HIV-1's obligate receptors. eCD4-Ig appears to be qualitatively different from antibodies, since it neutralizes all HIV-1, HIV-2, and SIV isolates. Here, we characterize three new structurally distinct eCD4-Ig variants and show that each excels in a key property useful to prevent, treat, or cure an HIV-1 infection. For example, one variant neutralized HIV-1 most efficiently, while others best enlisted natural killer cells to eliminate infected cells. These observations will help generate eCD4-Ig variants optimized for different clinical applications.

Antibody gene transfer for HIV immunoprophylaxis.

Antibody gene transfer, which involves the delivery of genes that encode potent, broadly neutralizing antibodies to human immunodeficiency virus (HIV), is a promising new strategy for preventing HIV infection. A satellite symposium at the AIDS Vaccine 2012 conference brought together many of the groups working in this field.

ICOS/B7RP-1 interference in mouse kidney transplantation.

BACKGROUND: Activated T cells play a key role in allograft rejection. T cell activation requires signaling via the T cell receptor as well as costimulatory signals. Inducible costimulatory molecule (ICOS), with its ligand B7RP-1, is a recently discovered costimulatory molecule of the CD28 family. The role of this signaling pathway during the early phases of kidney allograft rejection is not clear so far. METHODS: Kidneys were orthotopically transplanted from BALB/c to C57BL/6 mice. Animals were assigned to five experimental groups: blocking anti-ICOS monoclonal antibody, ICOS fusion protein, anti-B7RP1 monoclonal antibody, B7RP-1 fusion protein, and control immunoglobulin G. RESULTS: Survival was significantly reduced in animals treated with ICOS monoclonal antibody (mAb) and B7RP-1 Fc as compared with controls. These animals had also a lower number of apoptotic graft infiltrating T cells, whereas the expression of intracellular interferon-gamma in CD3CD4 T cells was increased. Animals treated with ICOS Fc and B7RP-1 mAb had similar survival and numbers of apoptotic T cells as compared with controls. CONCLUSIONS: In summary, the blockade of ICOS with ICOS mAb or B7RP-1 Fc reduced the amount of apoptosis of infiltrating lymphocytes and resulted in continuous inflammatory processes with progressive tissue damage and graft failure.

Terapia génica de la infección por el virus de la inmunodeficiencia humana. La moderna farmacoterapéutica del nuevo milenio / Gene therapy for HIV-1 infection, The modern pharmacotherapeutic strategies at the new millenium

La solución definitiva para la infección producida por el VIH-1 puede venir dada en el próximo milenio por la terapia génica. Guiados de sus objetivos mediante la utilización preferente de linfocitos maduros y células madre hematopoyéticas, las estrategias, algunas de ellas ya en ensayo clínico, se relacionan con el bloqueo de alguna de las etapas del ciclo vital del VIH. Así, podemos hablar de estrategias que se basan en: a) Acción a nivel de receptor/correceptor para el VIH (CD4/CCR-5); b) Transferencia de genes "suicidas" (timidina quinasa); c) Estimulación de la respuesta inmune contra el VIH (proteínas env/rev; receptor CD4-zeta); d) Transferencia de dominantes negativos (proteína RevM10), e) Utilización de ribozimas (anti-RNAs); f) Transferencia de anticuerpos contra proteínas vírales (anti-env); g) Utilización del propio VIH "furtivo" y, h) Protocolos específicos para las enfermedades asociadas. De cualquier forma, la aplicación de la terapia génica en el próximo milenio, posiblemente en su primer siglo, para el tratamiento de esa infección, vendrá dada por estrategias sinérgicas de forma combinada (AU)

Gene transfer of anti-gp41 antibody and CD4 immunoadhesin strongly reduces the HIV-1 load in humanized severe combined immunodeficient mice.

OBJECTIVE: To study the anti-HIV-1 effects of the delivery of anti-gp41 monoclonal antibody (mAb) and soluble CD4 (sCD4) immunoadhesin by genetically modified cells in HIV-1-infected, humanized severe combined immunodeficient (SCID) mice. DESIGN: The complementary DNA of mAb 2F5, an anti-HIV-1 gp41 antibody, and of sCD4-IgG chimeric immunoadhesin were transferred into 3T3 cells using Moloney murine leukaemia virus vectors. The cells were then incorporated into a collagen structure called the neo-organ, which allowed the continuous production of the therapeutic molecules. METHODS: The antiviral effects in vivo of 2F5 or sCD4-IgG or both compounds were evaluated in neo-organ-implanted SCID mice that were grafted with human CD4 CEM T cells and challenged with HIV-1 Lai or MN. RESULTS: In SCID mice implanted with 2F5 neo-organs, antibody plasma levels reached 500-2000 ng/ml. Viral loads after HIV-1 challenge were significantly reduced in neo-organ-implanted HIV-infected mice. Although 29 x 10(7) and 13 x 10(8) HIV-1-RNA copies/ml were detected at 12 days in the controls (mice injected with Lai and MN, respectively) less than 16.5 x 10(3) HIV-1-RNA copies/ml were observed in all implanted mice injected with either Lai or MN. The intracellular viral load was also reduced in CD4 cells recovered from the implanted mice. Comparable antiviral effects were obtained with CD4-IgG neo-organs. CONCLUSION: Our results confirm the anti-HIV properties of 2F5 and sCD4-IgG continuously produced in vivo after ex-vivo gene therapy in SCID mice.

Stable heterodimers from remodeling the domain interface of a homodimer using a phage display library.

Structure-guided phage display was used to select for combinations of interface residues for antibody C(H)3 domains that promote the formation of stable heterodimers. A C(H)3 "knob" mutant was made by replacement of a small residue, threonine, with a larger one, tryptophan: T366W. A library of C(H)3 "hole" mutants was then created by randomizing residues 366, 368 and 407, which are in proximity to the knob on the partner C(H)3 domain. The C(H)3 knob mutant was fused to a peptide flag and the C(H)3 hole library was fused to M13 gene III. Phage displaying stable C(H)3 heterodimers were recovered by panning using an anti-flag antibody. Phage-selected C(H)3 heterodimers differed in sequence from the previously designed heterodimer T366W-Y407'A, and most clones tested were more stable to guanidine hydrochloride denaturation. The thermal stability of individual C(H)3 domains secreted from Escherichia coli was analyzed by differential scanning calorimetry. One heterodimer, T366W-T366'S:L368'A:Y407'V, had a t(m) of 69.4 degrees C, which is 4.0 deg.C higher than that for the designed heterodimer and 11.0 deg.C lower than that for the wild-type homodimer. The phage-selected C(H)3 mutant maintained the preference for forming heterodimers over homodimers as judged by near-quantitative formation of an antibody/immunoadhesin hybrid in a cotransfection assay. Phage optimization provides a complementary and more comprehensive strategy to rational design for engineering homodimers for heterodimerization.

A bivalent immunoadhesin of the human interferon-gamma receptor is an effective inhibitor of IFN-gamma activity.

We describe here the bioengineering of a bivalent IFN-gamma-RFc immunoadhesin consisting of the extracellular domain of the human IFN-gamma receptor alpha chain (IFN-gamma-R) fused to a human IgG1 Fc region (encoding hinge, CH2 and CH3 domain) that was efficiently expressed as a covalently linked homodimer in insect cells and purified in a one-step purification procedure. The IFN-gamma-RFc fusion protein exerted a 3-fold higher ligand binding affinity in binding competition studies in vitro compared with the monovalent extracellular IFN-gamma-R domain. In addition, the in vitro antagonistic activity of IFN-gamma-RFc, as determined by inhibition of IFN-gamma-induced virus protection and HLA-DR expression, was more than 30-fold higher in comparison with the monovalent soluble receptor. The described IFN-gamma-R immunoadhesin is a potential therapeutic reagent to interfere with the disease-promoting activities of IFN-gamma in several autoimmune diseases.