Identification of overlapping but distinct cAMP and cGMP interaction sites with cyclic nucleotide phosphodiesterase 3A by site-directed mutagenesis and molecular modeling based on crystalline PDE4B

Cyclic nucleotide phosphodiesterase 3A (PDE3A) hydrolyzes cAMP to AMP, but is competitively inhibited by cGMP due to a low kcat despite a tight Km. Cyclic AMP elevation is known to inhibit all pathways of platelet activation, and thus regulation of PDE3 activity is significant. Although cGMP elevation will inhibit platelet function, the major action of cGMP in platelets is to elevate cAMP by inhibiting PDE3A. To investigate the molecular details of how cGMP, a similar but not identical molecule to cAMP, behaves as an inhibitor of PDE3A, we constructed a molecular model of the catalytic domain of PDE3A based on homology to the recently determined X-ray crystal structure of PDE4B. Based on the excellent fit of this model structure, we mutated nine amino acids in the putative catalytic cleft of PDE3A to alanine using site-directed mutagenesis. Six of the nine mutants (Y751A, H840A, D950A, F972A, Q975A, and F1004A) significantly decreased catalytic efficiency, and had kcat/Km less than 10% of the wild-type PDE3A using cAMP as substrate. Mutants N845A, F972A, and F1004A showed a 3- to 12-fold increase of Km for cAMP. Four mutants (Y751A, H840A, D950A, and F1004A) had a 9- to 200-fold increase of Ki for cGMP in comparison to the wild-type PDE3A. Studies of these mutants and our previous study identified two groups of amino acids: E866 and F1004 contribute commonly to both cAMP and cGMP interactions while N845, E971, and F972 residues are unique for cAMP and the residues Y751, H836, H840, and D950 interact with cGMP. Therefore, our results provide biochemical evidence that cGMP interacts with the active site residues differently from cAMP

Rational design of cytotoxic T-cell inhibitors

This study describes the use of the CD8/major histocompatibility complex (MHC) class I crystal structure as a template for the de novo design of low-molecular-weight surface mimetics. The analogs were designed from a local surface region on the CD8 a-chain directly adjacent to the bound MHC class I, to block the protein associations in the T-cell activation cluster that occur upon stimulation of the cytotoxic T lymphocytes (CTLs). One small conformationally restrained peptide showed dose-dependent inhibition of a primary allogeneic CTL assay while having no effect on the CD4-dependent mixed lymphocyte reaction (MLR). The analog\u27s activity could be modulated through subtle changes in its side chain composition. Administration of the analog prevented CD8-dependent clearance of a murine retrovirus in BALB/c mice. In C57BL/6 mice challenged with the same retrovirus, the analog selectively inhibited the antiviral CTL responses without affecting the ability of the CTLs to generate robust allogeneic responses

Adeno-Associated Virus Vector-Mediated Expression of Antirespiratory Syncytial Virus Antibody Prevents Infection in Mouse Airways.

Infants and older adults are especially vulnerable to infection by respiratory syncytial virus (RSV), which can cause significant illness and irreparable damage to the lower respiratory tract and for which an effective vaccine is not readily available. Palivizumab, a recombinant monoclonal antibody (mAb), is an approved therapeutic for RSV infection for use in high-risk infants only. Due to several logistical issues, including cost of goods and scale-up limitations, palivizumab is not approved for other populations that are vulnerable to severe RSV infections, such as older adults. In this study, we demonstrate that intranasal delivery of adeno-associated virus serotype 9 (AAV9) vector expressing palivizumab or motavizumab, a second-generation version of palivizumab, significantly reduced the viral load in the lungs of the BALB/c mouse model of RSV infection. Notably, we demonstrate that AAV9 vector-mediated prophylaxis against RSV was effective despite the presence of serum-circulating neutralizing AAV9 antibodies. These findings substantiate the feasibility of repeatedly administering AAV9 vector to the airway for seasonal prophylaxis against RSV, thereby expanding the application of vectored delivery of mAbs as an effective prophylaxis strategy against various airborne viruses

Adeno-Associated Virus 9-Mediated Airway Expression of Antibody Protects Old and Immunodeficient Mice against Influenza Virus

Influenza causes serious and sometimes fatal disease in individuals at risk due to advanced age or immunodeficiencies. Despite progress in the development of seasonal influenza vaccines, vaccine efficacy in elderly and immunocompromised individuals remains low. We recently developed a passive immunization strategy using an adeno-associated virus (AAV) vector to deliver a neutralizing anti-influenza antibody at the site of infection, the nasal airways. Here we show that young, old, and immunodeficient (severe combined immunodeficient [SCID]) mice that were treated intranasally with AAV9 vector expressing a modified version of the broadly neutralizing anti-influenza antibody FI6 were protected and exhibited no signs of disease following an intranasal challenge with the mouse-adapted H1N1 influenza strain A/Puerto Rico/8/1934(H1N1) (PR8) (Mt. Sinai strain). Nonvaccinated mice succumbed to the PR8 challenge due to severe weight loss. We propose that airway-directed AAV9 passive immunization against airborne infectious agents may be beneficial in elderly and immunocompromised patients, for whom there still exists an unmet need for effective vaccination against influenza

Localization of LacZ expression in injected muscle following IM injection of AAV8 in C57BL/6 mice.

<p>Localization of LacZ expression in injected muscle was determined at day 21 post-vector administration in C57BL/6 mice. Vector was administered at a dose of 10¹⁰ GC as two 25 µl injections into the right and left legs (A, B) or as one 2 µl injection (C, D). A lower dose of 10⁹ GC of vector was administered as two 25 µl injections into the right and left legs (E, F) or as one 2 µl injection (G, H). Three sections were taken throughout the injected gastrocnemius muscle of one representative animal per group (A, C, E and G) with one representative liver section per group (B, D, F and H) (n = 4/group).</p

Translation of influence of injection volume to a large animal model, the rhesus macaque.

<p>RAG KO mice were injected with 10¹⁰ GC AAV8.CMV.201Ig IA either by IM or IV injection; tissues were harvested on day 56 and analyzed for vector genome copies (GC), quantified as GC/diploid genome in (A) liver and (B) muscle. (C) Biodistribution of AAV8 vector was determined on day 90 post-vector administration in a rhesus macaque. Vector was administered at a dose of 3×10¹² GC/kg by IM injection into the vastus lateralis muscle of both right and left legs as 1 ml injections per kg body weight (vector concentration of 3×10¹² GC/ml). DNA and RNA were extracted for quantification of GC (open bars) and transcript levels of 201Ig IA (closed bars), respectively. Values for muscle are the average of measurements at 12 sites throughout the injected muscle and liver is the average of the four lobes, which were quantified separately. There was no detectable GC or RNA in control (un-injected) muscle samples. LN, lymph node; Ax, axillary; In, inguinal; Mes, mesenteric; Il, iliac; Pop, popliteal. (D) Time course of expression of 201Ig IA in serum. (E) Rhesus macaques were injected IM with 3×10¹¹ GC/kg of AAV8.CMV.201Ig IA, as either 1 ml vector injections per kg body weight (3×10¹¹ GC/ml) or 0.1 ml injection per kg body weight (3×10¹² GC/ml) (n = 2/group). Expression of 201Ig IA was measured in serum by ELISA and values are expressed as mean ± SEM. *<i>p</i><0.05.</p

Liver expression following IM vector administration in mice.

<p>Visualization of differential ffLuc expression patterns using Xenogen whole-body bioluminescent imaging on day 7 post-IM administration of 10¹⁰ GC AAV8.CMV.ffLuc (A) or AAV8.TBG.ffLuc (B) to C57BL/6 mice. Vector was administered as one 10 µl injection into the gastrocnemius muscle of the right leg. (C) ffLuc expression was quantified at day 28 post-vector administration by ffLuc tissue assays. ffLuc expression measured as RLU normalized to the total protein concentration of the organ. Data are presented as fold change over background where background was RLU/total protein of organ (g) in control tissues from un-injected mice. (D) Comparison of expression of 201Ig IA from the CMV and TBG promoters following a 10 µl IM injection in RAG KO mice. Expression of 201Ig IA in serum was measured by ELISA. Values are expressed as mean ± SEM (n = 4/group). ***<i>p</i><0.001.</p

Comparison of expression from the liver-specific TBG promoter following IM and IV vector administration in RAG KO mice.

<p>Expression on day 28 post-vector administration of 201Ig IA (A), 2.10A mAb (B), VRC01 mAb (C), and PG9 mAb (D) in RAG KO mice. Expression following IM injection of vectors with either the CMV or TBG promoter was compared to expression levels produced following IV injection of the TBG vector. Mice were injected IM with two 15 µl injections into the right and left gastrocnemius muscles. IV injections were performed as a 100 µl injection via the tail vein. Mice were administered with a dose of either 10¹⁰ GC or 10¹¹ GC, numbers indicate dose ×10¹¹ GC. ND, not determined. Expression was measured in serum by ELISA and values are expressed as mean ± SEM (n = 3/group). ***<i>p</i><0.001.</p