Trends in viral vector-based vaccines for tuberculosis: a patent review (2010–2023)

Abstract: Tuberculosis (TB) is an ancient global public health problem. Several strategies have been applied to develop new and more effective vaccines against TB, from attenuated or inactivated mycobacteria to recombinant subunit or genetic vaccines, including viral vectors. This review aimed to evaluate patents filed between 2010 and 2023 for TB vaccine candidates. It focuses on viral vectorbased strategies. A search was carried out in Espacenet, using the descriptors “mycobacterium and tuberculosis” and the classification A61K39. Of the 411 patents preliminarily identified, the majority were related to subunit vaccines, with 10 patents based on viral vector platforms selected in this study. Most of the identified patents belong to the United States or China, with a concentration of patent filings between 2013 and 2023. Adenoviruses were the most explored viral vectors, and the most common immunodominant Mycobacterium tuberculosis (Mtb) antigens were present in all the selected patents. The majority of patents were tested in mouse models by intranasal or subcutaneous route of immunization. In the coming years, an increased use of this platform for prophylactic and/or therapeutic approaches for TB and other diseases is expected. Along with this, expanding knowledge about the safety of this technology is essential to advance its use.

Safety and feasibility of the gene transfer of hemopexin for conditions with increased free heme.

Heme is a fundamental molecule for several biological processes, but when released in the extracellular space such as in hemolytic diseases, it can be toxic to cells and tissues. Hemopexin (HPX) is a circulating protein responsible for removing free heme from the circulation, whose levels can be severely depleted in conditions such as sickle cell diseases. Accordingly, increasing HPX levels represents an attractive strategy to mitigate the deleterious effects of heme in these conditions. Gene transfer of liver-produced proteins with adeno-associated virus (AAV) has been shown to be an effective and safety strategy in animal and human studies mainly in hemophilia. Here, we report the feasibility of increasing HPX levels using an AAV8 vector expressing human HPX (hHPX). C57Bl mice were injected with escalating doses of our vector, and expression was assessed by enzyme immunoassay (ELISA), Western blot, and quantitative polymerase chain reaction (qPCR). In addition, the biological activity of transgenic hHPX was confirmed using two different models of heme challenge consisting of serial heme injections or phenylhydrazine-induced hemolysis. Sustained expression of hHPX was confirmed for up to 26 weeks in plasma. Expression was dose-dependent and not associated with clinical signs of toxicity. hHPX levels were significantly reduced by heme infusions and phenylhydrazine-induced hemolysis. No clinical toxicity or laboratory signs of liver damage were observed in preliminary short-term heme challenge studies. Our results confirm that long-term expression of hHPX is feasible and safe in mice, even in the presence of heme overload. Additional studies are needed to explore the effect of transgenic HPX protein in animal models of chronic hemolysis.

Prevalence of Neutralizing Antibodies against Adeno-Associated Virus Serotypes 1, 2, and 9 in Non-Injected Latin American Patients with Heart Failure-ANVIAS Study.

Neutralizing antibody (NAb) activity against the viral capsid of adeno-associated viral (AAV) vectors decreases transduction efficiency, thus limiting transgene expression. Several reports have mentioned a variation in NAb prevalence according to age, AAV serotype, and, most importantly, geographic location. There are currently no reports specifically describing the anti-AAV NAb prevalence in Latin America. Here, we describe the prevalence of NAb against different serotypes of AAV vectors (AAV1, AAV2, and AAV9) in Colombian patients with heart failure (HF) (referred to as cases) and healthy individuals (referred to as controls). The levels of NAb were evaluated in serum samples of 60 subjects from each group using an in vitro inhibitory assay. The neutralizing titer was reported as the first dilution inhibiting ≥50% of the transgene signal, and the samples with neutralizing titers at ≥1:50 dilution were considered positive. The prevalence of NAb in the case and control groups were similar (AAV2: 43% and 45%, respectively; AAV1 33.3% in each group; AAV9: 20% and 23.2%, respectively). The presence of NAb for two or more of the serotypes analyzed was observed in 25% of the studied samples, with the largest amount in the positive samples for AAV1 (55-75%) and AAV9 (93%), suggesting serial exposures, cross-reactivity, or coinfection. Moreover, patients in the HF group exhibited more common combined seropositivity for NAb against AAV1 d AAV9 than those in the control group (91.6% vs. 35.7%, respectively; p = 0.003). Finally, exposure to toxins was significantly associated with the presence of NAb in all regression models. These results constitute the first report of the prevalence of NAb against AAV in Latin America, being the first step to implementing therapeutic strategies based on AAV vectors in this population in our region.

Development of a BoHV-4 viral vector expressing tgD of BoHV-1 and evaluation of its immunogenicity in mouse model.

In recent years, Bovine herpesvirus 4 (BoHV-4) has emerged as an attractive gene delivery viral vector, mainly for vaccination purposes in the veterinary field. In the present study, a new infectious clone of the BoHV-4 genome carrying a bacterial artificial chromosome vector (BoHV-4-BAC) was developed by homologous recombination in mammalian cell culture and bacterial systems, and exploited to express a truncated form of glycoprotein D (tgD) of Bovine herpesvirus 1 (BoHV-1) (BoHV-4-tgD∆TK) as a vaccine candidate. This construct's immunogenicity was compared to a DNA vector expressing the same antigen (pC-tgD) in a BALB/c mouse model. After the mice were immunized, total and specific antibody responses, cytokine responses, total splenocyte cells proliferation/cytotoxicity, and virus neutralization assays were conducted to analyze the immune response elicited by both constructs. Mice from both vaccine groups developed significant humoral and cellular immune responses after a booster dose regime was conducted on day 28 post-injection. In almost all immunological assays, BoHV-4-tgDΔTK induced as high an immune response as pC-tgD. In both vaccine constructs, neutralizing antibodies were a significant determining factor in protection against BoHV-1, even after the first injection. We conclude that a BoHV-4-based viral vector offers an effective immunization strategy as an alternative to DNA-based immunization platforms, at least to combat BoHV-1.

EXPRESIÓN DE LA PROTEÍNA CORE DEL VIRUS DE LA HEPATITIS C EN CÉLULAS HepG2 USANDO EL VIRUS DEL BOSQUE DE SEMLIKI / Hepatitis C virus core protein expression in HepG2 cells using Semliki Forest Virus

RESUMEN El Virus de la Hepatitis C (VHC) codifica la proteína Core. Que, además de ser la subunidad de la cápside, participa en diferentes mecanismos de patogénesis de la infección por VHC. Dado que el sistema de replicación in vitro del VHC presenta limitaciones, el uso de vectores virales podría ser una herramienta útil para estudiar las propiedades de la proteína Core. Con el fin de validar el vector con el Virus del Bosque de Semliki (SFV) para el estudio de Core en células HepG2, se evaluó la expresión de la proteína verde fluorescente (GFP) y la proteína Core utilizando este vector viral. Las expresiones de GFP y Core se detectaron en células HepG2 transducidas con rSFV de 24 a 96 horas postransducción. La expresión de la proteína Core fue inferior a la expresión de GFP en las células HepG2. Teniendo en cuenta que la proteína Core del VHC puede regular la actividad del gen p53, se evaluó el nivel transcripcional de este gen. Se observó una disminución en el nivel de mARN de p53 en las células luego de la transducción, comparado con las células control. Aunque las células transducidas con rSFV-Core presentaron el menor nivel de mARN de p53, la diferencia no fue significativa comparada con las células transducidas con rSFV-GFP. Los resultados confirman que rSFV permite la expresión transitoria de proteínas heterólogas en líneas celulares de hepatoma humano. Se necesitan estudios adicionales para determinar si la expresión disminuida de Core puede deberse a degradación de la proteína viral.

ABSTRACT The Hepatitis C Virus (HCV) encodes the structural protein Core, which in addition to being the capsid subunit, participates in different mechanisms of HCV infection pathogenesis. Since HCV in vitro replication system has limitations, the use of viral vectors could be a useful tool to study the Core protein properties. To validate the Semliki Forest Virus (SFV) strategy in transduced HepG2 cells to study the HCV Core protein, the expression of green fluorescent protein (GFP) and Core protein expressions were detected 24 to 96 hours post-transduction in HepG2 cells transduced with rSFV. Core protein expression was lower than GFP expression in HepG2 cells. Since HCV Core protein can regulate the activity of the p53 gene, the transcriptional level of this gene was evaluated. A decrease in the level of p53 mRNA was observed in the cells after transduction, compared to the control cells. Although the cells transduced with rSFV-Core had the lowest level of p53 mRNA, the difference was not significant compared to cells transduced with rSFV-GFP. The results confirm that rSFV allows the transient expression of heterologous proteins in human hepatoma cell lines. Additional studies are needed to determine whether the decreased expression of Core may be due to the degradation of the viral protein.

Lacrimal Gland as a Target Organ for Adenovirus Gene Therapy Encoding Erythropoietin for Dry Eye Induced by Benzalkonium Chloride.

Purpose: The aims of this work were a) to describe the histology of the lacrimal gland (LG) and cornea induced by an adenovirus (Ad) vector encoding the human erythropoietin (Epo) gene delivered to the LG and b) to evaluate the therapeutic potential of this strategy to prevent benzalkonium chloride (BAK) corneal toxicity.Methods: Structure and function of male Wistar rats LG were compared in the groups: 1) naïve control and 2) Ad-hEpo in the right LG (RLG). The protective response against BAK eye drops was compared among the groups 1) naïve control, 2) BAK in the right eye, 3) Ad-hEpo RLG + BAK and 4) Ad-hEpo in the right salivary gland (RSG)+BAK. Ad-hEpo groups received an injection of AdLTR2EF1a-hEPO (25 ul, 1010 particles/ml) in the right LG or SG (positive control). The BAK groups received 0.2% BAK in the right cornea twice a day. The tests applied after 7 days, included tear secretion, hEPO mRNA detection by qRT-PCR, LG and cornea histology, LG ELISA for cytokines and hematocrit.Results: hEPO mRNA was present in the Ad-hEpo RLG and RSG, but not kidney or liver samples (negative controls). TNF-α and IL-1ß increased in the LG exposed to Ad-hEpo compared to naïve control (p = .0115 and p = .0397, respectively). BAK reduced tear secretion, but this reduction was prevented by Ad-hEpo RLG+BAK and Ad-hEpo RSG+BAK (p = .017). The corneal epithelia were thinner in the BAK-treated groups independent of Ad-hEpo (p = .0009). Hematocrit increased only in the Ad-hEpo RSG group (p = .01).Conclusions: Ad-hEpo infection of rat LG and SG induces local, but only the SG infection induced systemic changes in rats. Importantly, Ad-hEpo attenuated the BAK-mediated toxic reduction in tear flow. Future studies must consider viral vector tissue tropism, biodistribution and effective therapeutic gene products for ocular surface diseases.

Vacunas para SARS-CoV-2: diferentes estrategias de los desarrollos en curso / Vaccines for SARS-CoV-2: different strategies of ongoing developments

El objetivo de este artículo es proporcionar una guía que sirva para la interpretación y seguimiento de los esfuerzos que se están desarrollando en todo el mundo con el objetivo de obtener una vacuna que pueda generar inmunidad contra el nuevo coronavirus SARS-CoV-2 de 2019, el agente causante de la enfermedad por coronavirus denominada COVID-19. Cinco meses después de haber sido detectada la enfermedad, ya hay 102 vacunas en distintos estadios de desarrollo, registradas por la Organización Mundial de la Salud (OMS), correspondientes a 8 plataformas vacunales con diferentes estrategias, y todos los días aparecen nuevas. Esto representará un enorme desafío de organismos internacionales, para la evaluación, comparación y selección de aquellas que cumplan con los criterios regulatorios indispensables de seguridad y eficacia y que, por otro lado, puedan ser producidas en cantidades suficientes para abastecer la demanda mundial. (AU)

The objective of this article is to provide a guide to help the interpretation and monitoring the efforts that are being carried out worldwide to obtain a vaccine that will be able to generate immunity against the new 2019 SARS-CoV-2 coronavirus, the viral agent causes the disease named COVID-19. Five months after the disease was detected, there are already 102 vaccines at different stages of development, registered by World Health Organization (WHO), corresponding to 8 vaccination platforms base on different strategies, and every day new ones appear. This will represent a huge challenge for international organizations, to evaluate, compare and selects those that will meet the essential regulatory criteria of safety and efficacy and that, would be able to be produced in enough quantities to supply the worldwide demand. Key words: SARS-Cov-2 vaccine, vaccine platform, COVID-19 strategy, attenuated virus, viral vector, viral proteins, viral DNA, viral RNA, nucleic acids, viral like particles, WHO. (AU)

Geographic shifts in the bioclimatic suitability for Aedes aegypti under climate change scenarios in Colombia.

The Dengue, Chikungunya and Zika viruses are arboviruses predominantly transmitted to humans through the bite of the female mosquito Aedes aegypti. Currently, the vector represents a potential epidemiological risk in several Latin American and Pacific countries. However, little is known about the geographical distribution and bioclimatic suitability of this mosquito in the projected climate change scenarios in Colombia. Using a species distribution model of maximum entropy (MaxEnt) based on presence-only records obtained from Global Biodiversity Information Facility (GBIF), land elevation obtained from Shuttle Radar Topography Mission (SRTM) and bioclimatic variables (WorldClim), we produced environmental suitability maps of this mosquito vector for present and future geographic distribution. The future distribution were constructed based on the Community Climate System Model (CCSM4) for the years 2050 and 2070, projected according to the Representative Concentration Pathways (RCP) 2.6, 4.5 and 8.5 described by the Intergovernmental Panel on Climate Change (IPCC). For the current conditions, Colombia has ~140,612.8 square km of areas with the possible presence of the vector; however, for the future, this will be reduced by more than 30%. For the future conditions, the suitable areas for A. aegypti decreased compared to the present, mainly for the year 2070 under RCP scenarios 4.5 and 8.5, however, the probability of mosquito occurrence increases in some departments of Colombia. Areas susceptible to the presence of A. aegypti are affected by climate change. The Caribbean and Andean regions have a high probability of mosquito distribution; therefore, control and epidemiological surveillance are required in these areas. The results can serve as an input to define preventive and control measures, especially in areas with a higher risk of contracting the virus.

[Immune response and gene therapy with adenoassociated viral vectors]. / Respuesta inmune y terapia génica con vectores virales adenoasociados.

In recent years, gene therapy has been positioned as a real and safe option in the development of therapeutic alternatives for the cure and prevention of different diseases. It consists in the insertion of genetic material in a defective tissue or cell, through the use of a vector. There are several considerations for selecting the most appropriate vector, including the potential for binding and entry to the target cell, the ability of the genetic material to transfer to the nucleus, the ability to express the insert, and the absence of toxicity. In the current scenario, the most commonly used viral vectors are those derived from adeno-associated viruses (AAV). Characteristics such as biosafety, low toxicity and selective tropism have enabled its evaluation as a therapeutic option in many monogenic or complex diseases. Despite their advantages, AAV vectors have drawbacks, the most important being the patient's immune response to the vector, especially the response mediated by neutralizing antibodies (NAb). NAbs decrease the transduction of the vector and prevent the expression of the gene it transports, limiting its clinical application. Therefore, identifying and quantifying the presence and activity of NAbs is the first step in any gene therapy protocol with AAV vectors. The presence of NAbs depends mainly on exposure to the virus in nature and varies drastically according to age, geographic location and health status of the person evaluated.

En los últimos años la terapia génica se ha posicionado como una opción real y segura en el desarrollo de alternativas terapéuticas para la cura y la prevención de diferentes enfermedades. Consiste en la inserción de material genético en un tejido o célula defectuosa, mediante el uso de un vector. Existen varias consideraciones para seleccionar el vector más apropiado, incluyendo el potencial de unión y entrada a la célula diana, la capacidad de transferencia del material genético al núcleo, la habilidad de expresión del inserto y la ausencia de toxicidad. En el panorama actual, los vectores virales más utilizados son los derivados de los virus adenoasociados (AAV). Características como su bioseguridad, baja toxicidad y tropismo selectivo, han posibilitado su evaluación como opción terapéutica en un amplio número de enfermedades monogénicas o complejas. A pesar de sus ventajas, los vectores AAV presentan inconvenientes, siendo el más importante la respuesta inmune del paciente al vector, especialmente la respuesta mediada por anticuerpos neutralizantes (NAb). Los NAb disminuyen la transducción del vector e impiden la expresión del gen que transporta, limitando su aplicación clínica. Por lo tanto, identificar y cuantificar la presencia y actividad de los NAbs, es el primer paso en cualquier protocolo de terapia génica con vectores AAV. La presencia de NAb depende principalmente de la exposición al virus en la naturaleza y varía drásticamente según edad, localización geográfica y estado de salud de la persona evaluada.

Ausencia de expresión de la proteína Core del virus de la hepatitis C en células dendríticas derivadas de monocitos humanos utilizando virus del Bosque de Semliki recombinante / Lack of expression of hepatitis c virus core protein in human monocyte-derived dendritic cells using recombinant semliki forest virus

ABSTRACT Hepatitis C Virus belongs to the Flaviviridae family. One proposed mechanism of HCV persistence in the ability to infect hematopoietic cells, including Dendritic cells (DCs). HCV infection of DCs could impair their functions that represent one of the mechanisms, thus hampering viral clearance by the host immune system. Among HCV-encoded proteins, the highly conserved Core protein has been suggested to be responsible for the immunomodulatory properties of this Hepacivirus. Recombinant viral vectors expressing the HCV Core protein and allowing its transduction and therefore the expression of the protein into DCs could be useful tools for the analysis of the properties of the Core protein. Vaccinia Virus and retrovirus have been used to transduce human DCs. Likewise, gene transfer into DCs using Semliki Forest Virus has been reported. This study aimed to express the HCV Core protein in human monocyte-derived DCs using an SFV vector, in which the subgenomic RNA encoding the structural proteins was replaced by the HCV Core sequence and then analyze the effects of its expression on DCs functions.

RESUMEN El virus de la Hepatitis C (VHC) pertenece a la familia Flaviviridae. Uno de los mecanismos propuestos de la persistencia del VHC es la capacidad de infectar células hematopoyéticas, incluidas las células dendríticas (DCs). La infección por VHC de DCs podría alterar sus funciones y corresponde a uno de los mecanismos que impiden el aclaramiento de la infección por VHC por el sistema inmunitario del hospedero. Entre las proteínas codificadas por el VHC, se ha sugerido que la proteína Core, altamente conservada, es responsable de las propiedades inmunomoduladoras de este Hepacivirus. Los vectores virales recombinantes que expresan la proteína Core y permiten su transducción a DCs podrían ser herramientas útiles para el análisis de las propiedades de esta proteína. El virus Vaccinia y el retrovirus se han utilizado para la transducción de DCs humanas. Del mismo modo, la transducción de DCs usando el virus del bosque de Semliki ha sido reportada. El objetivo de este estudio fue expresar la proteína Core de VHC en DCs derivadas de monocitos humanos utilizando un vector de SFV, en el que el ARN subgenómico que codifica las proteínas estructurales fue reemplazado por la secuencia Core del VHC y evaluar los efectos de su expresión en las funciones de DCs.

Respuesta inmune y terapia gó©nica con vectores virales adenoasociados / Immune response and gene therapy with adenoassociated viral vectors

En los ó;ºltimos aó;±os la terapia gó;©nica se ha posicionado como una opció;n real y segura en el desarrollo de alternativas terapó;©uticas para la cura y la prevenció;n de diferentes enfermedades. Consiste en la inserció;n de material genó;©tico en un tejido o có;©lula defectuosa, mediante el uso de un vector. Existen varias consideraciones para seleccionar el vector más apropiado, incluyendo el potencial de unió;n y entrada a la có;©lula diana, la capacidad de transferencia del material genó;©tico al nó;ºcleo, la habilidad de expresió;n del inserto y la ausencia de toxicidad. En el panorama actual, los vectores virales más utilizados son los derivados de los virus adenoasociados (AAV). Características como su bioseguridad, baja toxicidad y tropismo selectivo, han posibilitado su evaluació;n como opció;n terapó;©utica en un amplio nó;ºmero de enfermedades monogó;©nicas o complejas. A pesar de sus ventajas, los vectores AAV presentan inconvenientes, siendo el más importante la respuesta inmune del paciente al vector, especialmente la respuesta mediada por anticuerpos neutralizantes (NAb). Los NAb disminuyen la transducció;n del vector e impiden la expresió;n del gen que transporta, limitando su aplicació;n clínica. Por lo tanto, identificar y cuantificar la presencia y actividad de los NAbs, es el primer paso en cualquier protocolo de terapia gó;©nica con vectores AAV. La presencia de NAb depende principalmente de la exposició;n al virus en la naturaleza y varía drásticamente segó;ºn edad, localizació;n geográfica y estado de salud de la persona evaluada.

In recent years, gene therapy has been positioned as a real and safe option in the development of therapeutic alternatives for the cure and prevention of different diseases. It consists in the insertion of genetic material in a defective tissue or cell, through the use of a vector. There are several considerations for selecting the most appropriate vector, including the potential for binding and entry to the target cell, the ability of the genetic material to transfer to the nucleus, the ability to express the insert, and the absence of toxicity. In the current scenario, the most commonly used viral vectors are those derived from adeno-associated viruses (AAV). Characteristics such as biosafety, low toxicity and selective tropism have enabled its evaluation as a therapeutic option in many monogenic or complex diseases. Despite their advantages, AAV vectors have drawbacks, the most important being the patient’s immune response to the vector, especially the response mediated by neutralizing antibodies (NAb). NAbs decrease the transduction of the vector and prevent the expression of the gene it transports, limiting its clinical application. Therefore, identifying and quantifying the presence and activity of NAbs is the first step in any gene therapy protocol with AAV vectors. The presence of NAbs depends mainly on exposure to the virus in nature and varies drastically according to age, geographic location and health status of the person evaluated.

Advances in novel vaccines for foot and mouth disease: focus on recombinant empty capsids.

Foot and mouth disease (FMD) is a highly contagious disease of cloven-hoofed animals, which causes severe economic losses in the livestock industry. Currently available vaccines are based on inactivated FMD virus (FMDV). Although inactivated virus vaccines have proved to be effective in FMD control, they have a number of disadvantages, including the need for high bio-containment production facilities and the lack of induction of immunological memory. Novel FMD vaccines based on the use of recombinant empty capsids have shown promising results. These recombinant empty capsids are attractive candidates because they avoid the use of virus in the production facilities but conserve its complete repertoire of conformational epitopes. However, many of these recombinant empty capsids require time-consuming procedures that are difficult to scale up. Achieving production of a novel and efficient FMD vaccine requires not only immunogenic antigens, but also industrially relevant processes. This review intends to summarize and compare the different strategies already published for the production of FMDV recombinant empty capsids, focusing on large-scale production.

Development of a new tobamovirus-based viral vector for protein expression in plants.

Plants are becoming an interesting alternative system for the heterologous production of pharmaceutical proteins, providing a more scalable, cost-effective, and biologically safer option than the current expression systems. The development of plant virus expression vectors has allowed rapid and high-level transient expression of recombinant genes, and, in turn, provided an attractive plant-based production platform. Here we report the development of vectors based on the tobamovirus Pepper mild mottle virus (PMMoV) to be used in transient expression of foreign genes. In this PMMoV vector, a middle part of the viral coat protein gene was replaced by the green fluorescent protein (GFP) gene, and this recombinant genome was assembled in a binary vector suitable for plant agroinoculation. The accumulation of GFP was evaluated by observation of green fluorescent signals under UV light and by western blotting. Furthermore, by using this vector, the multiepitope gene for chikungunya virus was successfully expressed and confirmed by western blotting. This PMMoV-based vector represents an alternative system for a high-level production of heterologous protein in plants.

Determination of Anti-Adeno-Associated Viral Vector Neutralizing Antibodies in Patients With Heart Failure in the Cardiovascular Foundation of Colombia (ANVIAS): Study Protocol.

BACKGROUND: Recent progress in the pathophysiology of heart failure (HF) has led to the development of new therapeutic options such as gene therapy and the use of adeno-associated viral (AAV) vectors. Despite the promising results in early clinical trials of gene therapy for HF, various obstacles have been faced, such as the presence of neutralizing antibodies (NAbs) against the capsid vectors. NAb activity limits vector transduction levels and therefore diminishes the final therapeutic response. Recent studies evaluating the prevalence of NAbs in various populations found considerable geographic variability for each AAV serotype. However, the levels of NAbs in Latin American populations are unknown, becoming a limiting factor to conducting AAV vector therapeutic trials in this population. OBJECTIVE: The goal of this study is to determine for the first time, the prevalence of anti-AAV NAbs for the serotypes 1, 2, and 9 in HF patients from the city of Bucaramanga, Colombia, using the in vitro transduction inhibition assay. METHODS: We will conduct a cross-sectional study with patients who periodically attend the HF clinic of the Cardiovascular Foundation of Colombia and healthy volunteers matched for age and sex. For all participants, we will evaluate the NAb levels against serotypes AAV1, AAV2, and AAV9. We will determine NAb levels using the in vitro transduction inhibition assay. In addition, participants will answer a survey to evaluate their epidemiological and socioeconomic variables. Participation in the study will be voluntary and all participants will sign an informed consent document before any intervention. RESULTS: The project is in the first phase: elaboration of case report forms and the informed consent form, and design of the recruitment strategy. Patient recruitment is expected to begin in the spring of 2016. We expect to have preliminary results, including the titer of the viral vectors, multiplicity of infections that we will use for each serotype, and the general validation of the assay, at the end of 2016. The final results are expected mid-2017. CONCLUSIONS: This project is the first effort to evaluate NAb levels against AAV1, AAV2, and AAV9 serotypes in patients with HF in Latin America. Our results will allow us to check the cross-reactivity response between the serotypes assessed, to describe the epidemiological characteristics of the participant population, and to set up a link with earlier reports of NAb prevalence in the literature.

Safety of the novel influenza viral vector Brucella abortus vaccine in pregnant heifers / Segurança de uma nova vacina vetorial empregando o vírus influenza para Brucella abortus em novilhas prenhes

ABSTRACT: The present study provides the first information about the safety of a new influenza viral vector vaccine expressing the Brucella ribosomal protein L7/L12 or Omp16 containing the adjuvant Montanide Gel01 in pregnant heifers. Immunization of pregnant heifers was conducted via the conjunctival (n=10) or subcutaneous (n=10) route using cross prime and booster vaccination schedules at an interval of 28 days. The vector vaccine was evaluated in comparison with positive control groups vaccinated with B. abortus S19 (n=10) or B. abortus RB51 (n=10) and a negative (PBS+Montanide Gel01; n=10) control group. Clinical studies, thermometry, assessment of local reactogenicity and observation of abortion showed that the vector vaccine via the conjunctival or subcutaneous route was completely safe for pregnant heifers compared to the commercial vaccines B. abortus S19 or B. abortus RB51. The only single adverse event was the formation of infiltration at the site of subcutaneous injection; this reaction was not observed for the conjunctival route.

RESUMO: O presente estudo fornece as primeiras informações sobre a segurança de uma nova vacina usando o vetor viral influenza para expressar as proteínas de Brucella L7/L12 ou Omp16, contendo o adjuvante Montanide Gel01 em novilhas prenhes. A imunização de novilhas prenhes foi realizada através das vias conjuntiva (n=10) ou subcutânea (n=10), empregadas na primovacinação e na dose de reforço. O intervalo foi de 28 dias. A vacina empregando o vetor foi comparada com os grupos de controle positivo, vacinados com B. abortus B19 (n=10) ou B. abortus RB51 (n=10) e um grupo de controle negativo (PBS + Montanide Gel01; n=10). Os estudos clínicos, termometria, reação local e observação do aborto mostraram que a vacina empregando o vetor, aplicada pela via conjuntival ou subcutânea, foi completamente segura para novilhas prenhes, em comparação com as vacinas comerciais B. abortus B19 ou B. abortus RB51. O único efeito adverso foi a formação de infiltrado no local da administração subcutânea; essa reação não foi observada no grupo vacinado pela via conjuntival.

Safety of the novel influenza viral vector Brucella abortus vaccine in pregnant heifers

ABSTRACT: The present study provides the first information about the safety of a new influenza viral vector vaccine expressing the Brucella ribosomal protein L7/L12 or Omp16 containing the adjuvant Montanide Gel01 in pregnant heifers. Immunization of pregnant heifers was conducted via the conjunctival (n=10) or subcutaneous (n=10) route using cross prime and booster vaccination schedules at an interval of 28 days. The vector vaccine was evaluated in comparison with positive control groups vaccinated with B. abortus S19 (n=10) or B. abortus RB51 (n=10) and a negative (PBS+Montanide Gel01; n=10) control group. Clinical studies, thermometry, assessment of local reactogenicity and observation of abortion showed that the vector vaccine via the conjunctival or subcutaneous route was completely safe for pregnant heifers compared to the commercial vaccines B. abortus S19 or B. abortus RB51. The only single adverse event was the formation of infiltration at the site of subcutaneous injection; this reaction was not observed for the conjunctival route.

RESUMO: O presente estudo fornece as primeiras informações sobre a segurança de uma nova vacina usando o vetor viral influenza para expressar as proteínas de Brucella L7/L12 ou Omp16, contendo o adjuvante Montanide Gel01 em novilhas prenhes. A imunização de novilhas prenhes foi realizada através das vias conjuntiva (n=10) ou subcutânea (n=10), empregadas na primovacinação e na dose de reforço. O intervalo foi de 28 dias. A vacina empregando o vetor foi comparada com os grupos de controle positivo, vacinados com B. abortus B19 (n=10) ou B. abortus RB51 (n=10) e um grupo de controle negativo (PBS + Montanide Gel01; n=10). Os estudos clínicos, termometria, reação local e observação do aborto mostraram que a vacina empregando o vetor, aplicada pela via conjuntival ou subcutânea, foi completamente segura para novilhas prenhes, em comparação com as vacinas comerciais B. abortus B19 ou B. abortus RB51. O único efeito adverso foi a formação de infiltrado no local da administração subcutânea; essa reação não foi observada no grupo vacinado pela via conjuntival.

Safety of the novel influenza viral vector Brucella abortus vaccine in pregnant heifers

ABSTRACT: The present study provides the first information about the safety of a new influenza viral vector vaccine expressing the Brucella ribosomal protein L7/L12 or Omp16 containing the adjuvant Montanide Gel01 in pregnant heifers. Immunization of pregnant heifers was conducted via the conjunctival (n=10) or subcutaneous (n=10) route using cross prime and booster vaccination schedules at an interval of 28 days. The vector vaccine was evaluated in comparison with positive control groups vaccinated with B. abortus S19 (n=10) or B. abortus RB51 (n=10) and a negative (PBS+Montanide Gel01; n=10) control group. Clinical studies, thermometry, assessment of local reactogenicity and observation of abortion showed that the vector vaccine via the conjunctival or subcutaneous route was completely safe for pregnant heifers compared to the commercial vaccines B. abortus S19 or B. abortus RB51. The only single adverse event was the formation of infiltration at the site of subcutaneous injection; this reaction was not observed for the conjunctival route.

RESUMO: O presente estudo fornece as primeiras informações sobre a segurança de uma nova vacina usando o vetor viral influenza para expressar as proteínas de Brucella L7/L12 ou Omp16, contendo o adjuvante Montanide Gel01 em novilhas prenhes. A imunização de novilhas prenhes foi realizada através das vias conjuntiva (n=10) ou subcutânea (n=10), empregadas na primovacinação e na dose de reforço. O intervalo foi de 28 dias. A vacina empregando o vetor foi comparada com os grupos de controle positivo, vacinados com B. abortus B19 (n=10) ou B. abortus RB51 (n=10) e um grupo de controle negativo (PBS + Montanide Gel01; n=10). Os estudos clínicos, termometria, reação local e observação do aborto mostraram que a vacina empregando o vetor, aplicada pela via conjuntival ou subcutânea, foi completamente segura para novilhas prenhes, em comparação com as vacinas comerciais B. abortus B19 ou B. abortus RB51. O único efeito adverso foi a formação de infiltrado no local da administração subcutânea; essa reação não foi observada no grupo vacinado pela via conjuntival.

Safety of the novel influenza viral vector Brucella abortus vaccine in pregnant heifers / Segurança de uma nova vacina vetorial empregando o vírus influenza para Brucella abortus em novilhas prenhes

The present study provides the first information about the safety of a new influenza viral vector vaccine expressing the Brucella ribosomal protein L7/L12 or Omp16 containing the adjuvant Montanide Gel01 in pregnant heifers. Immunization of pregnant heifers was conducted via the conjunctival (n=10) or subcutaneous (n=10) route using cross prime and booster vaccination schedules at an interval of 28 days. The vector vaccine was evaluated in comparison with positive control groups vaccinated with B. abortus S19 (n=10) or B. abortus RB51 (n=10) and a negative (PBS+Montanide Gel01; n=10) control group. Clinical studies, thermometry, assessment of local reactogenicity and observation of abortion showed that the vector vaccine via the conjunctival or subcutaneous route was completely safe for pregnant heifers compared to the commercial vaccines B. abortus S19 or B. abortus RB51. The only single adverse event was the formation of infiltration at the site of subcutaneous injection; this reaction was not observed for the conjunctival route.(AU)

O presente estudo fornece as primeiras informações sobre a segurança de uma nova vacina usando o vetor viral influenza para expressar as proteínas de Brucella L7/L12 ou Omp16, contendo o adjuvante Montanide Gel01 em novilhas prenhes. A imunização de novilhas prenhes foi realizada através das vias conjuntiva (n=10) ou subcutânea (n=10), empregadas na primovacinação e na dose de reforço. O intervalo foi de 28 dias. A vacina empregando o vetor foi comparada com os grupos de controle positivo, vacinados com B. abortus B19 (n=10) ou B. abortus RB51 (n=10) e um grupo de controle negativo (PBS + Montanide Gel01; n=10). Os estudos clínicos, termometria, reação local e observação do aborto mostraram que a vacina empregando o vetor, aplicada pela via conjuntival ou subcutânea, foi completamente segura para novilhas prenhes, em comparação com as vacinas comerciais B. abortus B19 ou B. abortus RB51. O único efeito adverso foi a formação de infiltrado no local da administração subcutânea; essa reação não foi observada no grupo vacinado pela via conjuntival.(AU)

Gene therapy for cardiovascular disease: advances in vector development, targeting, and delivery for clinical translation.

Gene therapy is a promising modality for the treatment of inherited and acquired cardiovascular diseases. The identification of the molecular pathways involved in the pathophysiology of heart failure and other associated cardiac diseases led to encouraging preclinical gene therapy studies in small and large animal models. However, the initial clinical results yielded only modest or no improvement in clinical endpoints. The presence of neutralizing antibodies and cellular immune responses directed against the viral vector and/or the gene-modified cells, the insufficient gene expression levels, and the limited gene transduction efficiencies accounted for the overall limited clinical improvements. Nevertheless, further improvements of the gene delivery technology and a better understanding of the underlying biology fostered renewed interest in gene therapy for heart failure. In particular, improved vectors based on emerging cardiotropic serotypes of the adeno-associated viral vector (AAV) are particularly well suited to coax expression of therapeutic genes in the heart. This led to new clinical trials based on the delivery of the sarcoplasmic reticulum Ca(2+)-ATPase protein (SERCA2a). Though the first clinical results were encouraging, a recent Phase IIb trial did not confirm the beneficial clinical outcomes that were initially reported. New approaches based on S100A1 and adenylate cyclase 6 are also being considered for clinical applications. Emerging paradigms based on the use of miRNA regulation or CRISPR/Cas9-based genome engineering open new therapeutic perspectives for treating cardiovascular diseases by gene therapy. Nevertheless, the continuous improvement of cardiac gene delivery is needed to allow the use of safer and more effective vector doses, ultimately bringing gene therapy for heart failure one step closer to reality.