Codon-Optimized and de novo-Synthesized E-Selectin/AAV2 Dose-Response Study for Vascular Regeneration Gene Therapy.

OBJECTIVE: This study focuses on dose-response investigation using a codon-optimized and de novo-synthesized E-Selectin/AAV2 (E-Sel/AAV2) vector in preparation for Investigational New Drug enabling of subsequent clinical studies. BACKGROUND: Gene therapy is a potential solution for patients suffering from chronic limb-threatening ischemia. Understanding the dose for effective gene delivery is crucial for future Investigational New Drug-enabling studies. METHODS: Expression of the codon-optimized E-Selectin gene was assessed by flow cytometry following in vitro cell transfection assay and RT-qPCR for murine limbs injected in vivo with AAV-m-E-Selectin (E-Sel/AAV2). Dose-response studies involved 3 cohorts of FVB/NJ mice (n=6/group) with escalating log doses of E-Selectin/AAV2 injected intramuscularly in divided aliquots, ranging from 2 × 10 9 VG to 2 × 10 11 VG, into ischemic limbs created by left femoral artery/vein ligation/excision and administration of nitric oxide synthase inhibitor, L-NAME. Limb perfusion, extent of gangrene free limb, functional limb recovery, and therapeutic angiogenesis were assessed. RESULTS: Codon-optimized E-Sel/AAV2 gene therapy exhibits a superior expression level than WT E-Sel/AAV2 gene therapy both in vitro and in vivo. Mice treated with a high dose (2 × 10 11 VG) of E-Sel/AAV2 showed significantly improved perfusion indices, lower Faber scores, increased running stamina, and neovascularization compared with lower doses tested with control groups, indicating a distinct dose-dependent response. No toxicity was detected in any of the animal groups studied. CONCLUSIONS: E-Sel/AAV2 Vascular Regeneration Gene Therapy holds promise for enhancing the recovery of ischemic hindlimb perfusion and function, with the effective dose identified in this study as 2 × 10 11 VG aliquots injected intramuscularly.

Antigenicidad e inmunogenicidad de una cepa inactivada de Vibrio cholerae O1 El Tor Inaba / Antigenicity and immunogenicity of inactivated Vibrio cholerae 01 El Tor Inaba

INTRODUCCIÓN: el cólera continúa siendo problema de salud, principalmente en las zonas más pobres. La mayoría de los aislamientos que se realizan al nivel mundial pertenecen a Vibrio cholerae O1 biotipo El Tor serotipo Ogawa, aunque se han reportado brotes causados por el serotipo Inaba, por lo que se aconseja que una vacuna efectiva contra la enfermedad deberá contener células o antígenos de ambos serotipos. OBJETIVO: describir las características de la cepa de V. cholerae O1 El Tor Inaba C6706 como posible cepa para la obtención de vacunas inactivadas contra el cólera. MÉTODOS: se elaboró un lote de siembra de trabajo crioconservado a - 70 ºC, se evaluó su identidad, pureza y viabilidad. La cepa fue cultivada en caldo triptona soya y su inactivación se realizó a 56 ºC durante 20 min. Se evaluó la capacidad antigénica del producto obtenido mediante western blot y ELISA de inhibición de lipopolisacárido. La inmunogenicidad en conejos, inoculados por vía intraduodenal, se determinó mediante la cinética de anticuerpos vibriocidas. RESULTADOS: el lote de siembra de trabajo mantuvo su identidad, pureza y viabilidad durante 18 meses de estudio. Se observó la presencia de los antígenos lipopolisacárido, hemaglutinina sensible a manosa y proteína de membrana externa U, en las suspensiones de células inactivadas de V. cholerae O1, no así de las subunidades A y B de la toxina colérica (CTA y CTB, respectivamente) o pilli corregulado con la toxina (TCP). Por otra parte, se obtuvieron títulos vibriocidas en los sueros de conejos, similares a la respuesta inducida por la cepa viva atenuada 638 de V. cholerae O1, candidata a vacuna oral. CONCLUSIONES: la cepa de V. cholerae O1 El Tor Inaba C6706 mostró características culturales, antigénicas e inmunogénicas que permiten considerarla como posible cepa para la obtención de vacunas inactivadas contra el cólera.

INTRODUCTION: cholera continues being a serius health problem, mainly in the poorest areas. Most of the isolations worldwide belong to Vibrio cholerae O1 biotype El Tor serotype Ogawa, although there have been reported outbreaks caused by serotype Inaba, that is why it is recommended that an effective vaccine against the illness should contain cells or antigens of both serotypes. OBJECTIVE: to describe the characteristics of the strain C6706 of V. cholerae O1 El Tor Inaba as a likely strain for the obtaining of inactivated vaccines for cholera. METHODS: a criopreserved working culture batch was made up at - 70 ºC to evaluate identity, purity and viability. The strain was cultured in tryptone soy broth and was inactivated at 56º C for 20 minutes. Antigenicity of this preparation was tested by Western Blot and ELISA inhibition test. Immunogenicity in adult rabbits, inoculated intraduodenally, was determined by means of kinetics of vibriocidal antibodies. RESULTS: the working culture batch kept their identity, purity and viability during 18 months of study. The results confirmed the presence of relevant antigens as lipopolysaccharide (LPS), mannose-sensitive hemagglutinin (MSHA) and outer membrane protein U (OmpU) in the suspension of inactivated V. cholerae O1 cells, but not cholera toxin subunits (CTA, CTB) or toxin-coregulated pili (TCP).On the other hand, vibriocidal titers were found in rabbit sera, which were similar to those previously reported for the live V. cholerae O1 strain 638, an attenuated oral vaccinal candidate CONCLUSIONS: the V. cholerae O1 El Tor Inaba C6706 strain showed cultural, antigenic and immunogenic characteristics that allow us to consider it as a possible strain for the obtaining of inactivated vaccines for cholera.

Passive protection with immunoglobulin A antibodies against tuberculous early infection of the lungs.

We report on a new approach toward protection against tuberculosis, based on passive inoculation with immunoglobulin A (IgA) antibodies. In a mouse model of tuberculous lung infection, intranasal inoculations of mice with an IgA monoclonal antibody (mAb) against the alpha-crystallin antigen of Mycobacterium tuberculosis reduced up to 10-fold the lung bacterial counts at nine days after either aerosol- or intranasal challenge. This effect involved synergism between mAb inoculations shortly before and 3 days after infection. Monomeric IgA reduced the colony-forming unit counts to the same extent as the polymeric IgA, suggesting antibody targeting to Fcalpha, rather than poly-immunoglobulin receptors on infected lung macrophages. The protective effect was of short duration, presumably due to the rapid degradation of the intranasally applied IgA. Our results provide evidence of an alternative approach which could be further developed toward immunoprophylaxis against tuberculosis in immunocompromised subjects.