Human Serotype Research Articles

Clinicopathological Study of Seronegative Celiac Disease in Adults in Pakistan: A Pilot Study.

BACKGROUND Celiac disease (CD) is usually missed, if the serology is negative. We aimed to evaluate the clinicopathological characteristics of seronegative CD (SNCD) and its response to gluten-free diet (GFD) in adult patients. METHODS This observational study was carried out at the Department of Hepatogastroenterology, Sindh Institute of Urology and Transplantation, Karachi, Pakistan from 2009 to 2015. All consecutive adult patients (≥17 years) with features of marked villous atrophy (Marsh class≥III) on duodenal biopsy, negative tissue transglutaminase IgA and IgG antibodies (anti-tTg IgA and IgG) and human leukocyte antigen (HLA) DQ2 or DQ8 serotypes were studied. Clinical characteristics, laboratory parameters, and response to GFD were analyzed by SPSS software version 20. Median and interquartile range (IQR) were used for summarizing quantitative data. Frequency (percentages) was used for qualitative data. RESULTS A total of 12 patients with median age of 31.5 years (IQR: 19.75-46.75 years), of whom five (41.6%) were men were studied. The presenting complaints were: weight loss in 11 (91.6%) and abdominal pain in 9 (75%) patients. Anemia was observed in 10 (83.3%) patients with median hemoglobin of 9.5 g/dL (IQR: 6.3-13.25 g/dL). Median alanine transaminase (ALT) was 21 U/L (IQR: 13–27 U/L) and median albumin was 3 g/dL (IQR: 2.4-3.6 g/dL). Anti-tTg IgA and IgG were negative in all patients. HLA DQ serotyping showed homozygous DQ2 and DQ8 in four and one patients, respectively; while heterozygous DQ2 and DQ8 in five and two patients, respectively. All patients were advised to receive GFD. Nine (75%) patients showed complete clinical response. Two patients were non-compliant and one with non-alcoholic fatty liver disease (NAFLD)-related cirrhosis had partial clinical response. Out of the nine responders, two patients showed response within 6 months while the remaining showed improvement over a year period. CONCLUSION The diagnosis of SNCD is rewarding as it responds favorably to GFD in most patients. HLA serology provides an important tool for diagnosis of this entity.

High Prevalence of Infectious Adeno-associated Virus (AAV) in Human Peripheral Blood Mononuclear Cells Indicative of T Lymphocytes as Sites of AAV Persistence

Adeno-associated virus is viewed as apathogenic and replication defective, requiring coinfection with adenovirus or herpesvirus for productive infection. In vivo persistence of a defective virus requires latency in specialized cell types to escape the host immune response until viral spread becomes possible. Reactivation from latency can be induced by diverse stimuli, including infections, typically induced upon host immunosuppression. We show for the first time that infectious AAV is highly prevalent in human leukocytes, specifically T lymphocytes, and that AAV is strongly amplified upon immunosuppression, along with reactivation of latent human herpesviruses. In the absence of an animal model to study the AAV life cycle, our findings in the human host will advance the understanding of AAV latency, reactivation, and in vivo pathogenesis.

Cell-Surface Integrins and CAR Are Both Essential for Adenovirus Type 5 Transduction of Canine Cells of Lymphocytic Origin.

Adenoviruses are the most widely used vectors in cancer gene therapy. Adenoviruses vectors are well characterized and are easily manipulated. Adenovirus serotype 5 (Ad5) is the most commonly used human serotype. Ad5 internalization into host cells is a combined effect of binding of Ad5 fiber knob with the coxsackie virus and adenovirus receptor (CAR) and binding of RGD motifs in viral penton to cell surface integrins (αvβ3, αvβ5). Ad5’s wide range of host-cell transduction and lack of integration into the host genome have made it an excellent choice for cancer therapeutics. However, Ad5 has limited ability to transduce cells of hematopoietic origin. It has been previously reported that low or no expression of CAR is a potential obstacle to Ad5 infection in hematopoietic origin cells. In addition, we have previously reported that low levels of cell surface integrins (αvβ3, αvβ5) may inhibit Ad5 infection in canine lymphoma cell lines. In the current report we have examined the ability of an Ad5 vector to infect human (HEK293) and canine non-cancerous (NCF and PBMC), canine non-hematopoietic origin cancer (CMT28, CML7, and CML10), and canine hematopoietic origin cancer (DH82, 17–71, OSW, MPT-1, and BR) cells. In addition, we have quantified CAR, αvβ3 and αvβ5 integrin transcript expression in these cells by using quantitative reverse transcriptase PCR (q-RT-PCR). Low levels of integrins were present in MPT1, 17–71, OSW, and PBMC cells in comparison to CMT28, DH82, and BR cells. CAR mRNA levels were comparatively higher in MPT1, 17–71, OSW, and PBMC cells. This report confirms and expands the finding that low or absent expression of cell surface integrins may be the primary reason for the inability of Ad5-based vectors to transduce cells of lymphocytic origin and some myeloid cells but this is not true for all hematopoietic origin cells. For efficient use of Ad5-based therapeutic vectors in cancers of lymphocytic origin, it is important to address the defects in cell surface integrins.

Ribonuclease from Bacillus Acts as an Antiviral Agent against Negative- and Positive-Sense Single Stranded Human Respiratory RNA Viruses.

Bacillus pumilus ribonuclease (binase) was shown to be a promising antiviral agent in animal models and cell cultures. However, the mode of its antiviral action remains unknown. To assess the binase effect on intracellular viral RNA we have selected single stranded negative- and positive-sense RNA viruses, influenza virus, and rhinovirus, respectively, which annually cause respiratory illnesses and are characterized by high contagious nature, mutation rate, and antigen variability. We have shown that binase exerts an antiviral effect on both viruses at the same concentration, which does not alter the spectrum of A549 cellular proteins and expression of housekeeping genes. The titers of influenza A (H1N1pdm) virus and human rhinovirus serotype 1A were reduced by 40% and 65%, respectively. A preincubation of influenza virus with binase before infection significantly reduced viral titer after single-cycle replication of the virus. Using influenza A virus mini genome system we showed that binase reduced GFP reporter signaling indicating a binase action on the expression of viral mRNA. Binase reduced the level of H1N1pdm viral NP mRNA accumulation in A549 cells by 20%. Since the viral mRNA is a possible target for binase this agent could be potentially applied in the antiviral therapy against both negative- and positive-sense RNA viruses.

Human avian influenza virus serotypes in a tertiary hospital in Nigeria

Background: Nigeria suffered waves of Highly Pathog enic Avian Influenza (HPAI) outbreaks that peaked twice in February 2006 and February 2007. The outbr eaks affected 3,057 commercial and rural household farms causing 1.3 million of the country’ s poultry destroyed. The burden of Influenza is likely to be under estimated and because of the uns table nature of the virus it call for the mapping o f this virus to specific region, this study therefore seeks to identify the types, prevalence, strain, predilection of the organism so as to monitor the o ccurrence of influenza in this part of the country with the aim of providing a base line level of intervent ion of influenza virus and find the candidate virus for vaccine selection and production. This study is a c ross sectional survey, our target populations were all pediatric and adult patients admitted at the pe diatrics and internal medicine department of ATBUTH who has met the criteria for presence of cough, fev er (>37 o C), nasal congestion and dyspnoea. Samples were collected and analyzed using the polym erase chain reaction (PCR), for the presence and sub typing of influenza viruses in respiratory spec imens. Overall 5% of the samples collected tested positive for human influenza type A and B. During a verage epidemics, overall attack rates are estimate d to be 10–20%, but in certain susceptible population s such as schoolchildren or nursing home residents, attack rates of 40–50% may occur. 60% of the positi ve results were among the female samples where as 40% from the male samples

Development of Novel Vaccines Against Infectious Diseases Based on Chimpanzee Adenoviral Vector.

Vaccination is considered to be the most effective method of preventing infectious or other diseases. Adenovirus (Ad) is one the most promising vectors in vaccine research and development. It can induce not only potent humoral but also cellular immune responses, and has therefore been widely applied in basic and translational studies. Chimpanzee Ad is a rare serotype circulating in humans. This circumvents the problem of preexisting immunity to human Ad serotypes, enhancing Chimpanzee Ad prospects in vaccine development. Here we describe experimental procedures used to generate a new generation of rabies vaccine based on a chimpanzee Ad vector, which can be extended in the development of novel vaccines against other infectious diseases.

Review on Adeno Virus; As a Vaccine Vehicle

Adenoviruses have moved to the forefront of vaccinology and are showing substantial prom ise as vehicles for antigen delivery for a number of vaccines currently being developed. Most studies to date have focused on human serotype adenoviruses, particularly human adenovirus type 5. Human serotype adenovirus vaccine vectors are particularly usef ul for development of veterinary vaccines as neutralizing antibodies to the vector will not usually be present in the vaccinates. Most vectors currently used as vaccine carriers are deleted in E1 gene. The original E1 deleted adenoviral vectors were constr ucted by homologous recombination. Replication incompetent vectors contain an antigen expression cassette substituted for the deleted E1A – E1B region. These replication incompitant adenoviruses can not replicate because of the deletion of the essential vir al E1 gene region containing two genes. Replication competent adenoviral vectors encode all of the remaining adenoviral antigens in addition to the transgene product, i.e., the vaccine antigen. The potential for adenoviruses to elicit powerful B cell and T cell responses in the mammalian host are the main reason for the use of these vectors in vaccine development. For effective veterinary use, extensive research on adenoviral vaccine vectors should be undertaken.

Antitumor effects of recombinant human adenovirus-p53 against human cutaneous squamous cell carcinoma in mice.

The present study was conducted to identify the anti-tumor effects of rAd/p53, which is a recombinant human serotype 5 adenovirus, in cutaneous squamous cell carcinoma (cSCC). Mouse models of human cSCC were constructed by injecting human cutaneous squamous cell carcinoma cells into both flanks of nude mice. Subsequently, the 75 nude mice with cSCC xenograft tumors were randomly divided into recombinant human serotype 5 adenovirus (rAd)/p53, rAd/p53 + 5-fluorouracil (5-Fu) and 5-Fu groups. One side of the tumors was administered the therapeutic agents as the therapeutic group, whereas the remaining side was treated with medical saline as the control. At 24, 48, 72, 120 and 168 h post-intratumoral injection, alterations in tumor volume, tumor necrosis and the expression of several tumor-associated genes, including Smad4, Brca1 and matrix metalloproteinase (MMP-2), were analyzed. Compared with its control group, the rAd/P53 group exhibited a significantly increased tumor necrosis ratio. In addition, Smad4 and Brca1 expression levels increased significantly at various time points (P<0.05), and MMP-2 expression decreased significantly (P<0.05). In the rAd/p53 + 5-Fu group, the tumor necrosis ratio, and Smad4 and Brca1 expression levels also significantly increased at various time points (P<0.05). MMP-2 gene transcription gradually decreased, high expression of Smad4 was prolonged, and high expression of Brca1 was observed in the early period following treatment compared with the rAd/P53 group. In addition, p53 expression exhibited a positive correlation with the tumor necrosis ratio and Smad4 expression, and showed a negative correlation with MMP-2 gene transcription (P<0.05). These findings indicate that rAd/p53 has a potent anti-tumor effect in cSCC via the promotion of tumor necrosis and regulating the expression of various tumor-associated genes.

Prevalence and 3-year persistence of human papillomavirus serotypes in asymptomatic patients in Northern Mexico.

To investigate clinical outcomes and 3-year persistence of human papillomavirus (HPV) infections among women in Mexico. A prospective study enrolled sexually active women attending primary healthcare clinics in metropolitan Monterrey, Mexico, between June 3 and August 30, 2002. Baseline data were collected and participants underwent HPV screening. Patients with HPV infections were asked to attend a repeat screening appointment after 3years, when the same screening data were gathered. Descriptive analyses were performed and the prevalence of cervical lesions and viral infections were examined. In total, 1188 patients who underwent initial HPV screening were included. Cervical lesions were detected in 5 (0.4%) patients and 239 (20.1%) patients had HPV infections; 129 (54.0%) of these patients attended 3-year follow-up. Among the 357 HPV serotypes identified, the most prevalent serotypes were HPV-59, HPV-52, HPV-16, and HPV-56, detected 62 (17.4%), 38 (10.6%), 27 (7.6%), and 18 (5.0%) times, respectively. Of the 129 patients attending 3-year follow-up, 104 (80.6%) were clear from HPV infections, 13 (10.1%) patients had persistent HPV infections, and 12 (9.3%) had HPV infections with different HPV types. The HPV prevalence was 20.1% in the present study; the most prevalent infections were HPV-59, HPV-52, HPV-16, and HPV-56. At 3-year follow-up, 25 (19.4%) patients had HPV infections.

Oral Administration of Astrovirus Capsid Protein Is Sufficient To Induce Acute Diarrhea In Vivo.

ABSTRACTThe disease mechanisms associated with the onset of astrovirus diarrhea are unknown. Unlike other enteric virus infections, astrovirus infection is not associated with an inflammatory response or cellular damage. In vitro studies in differentiated Caco-2 cells demonstrated that human astrovirus serotype 1 (HAstV-1) capsid protein alone disrupts the actin cytoskeleton and tight junction complex, leading to increased epithelial barrier permeability. In this study, we show that oral administration of purified recombinant turkey astrovirus 2 (TAstV-2) capsid protein results in acute diarrhea in a dose- and time-dependent manner in turkey poults. Similarly to that induced by infectious virus, TAstV-2 capsid-induced diarrhea was independent of inflammation or histological changes but was associated with increased intestinal barrier permeability, as well as redistribution of sodium hydrogen exchanger 3 (NHE3) from the membrane to the cytoplasm of the intestinal epithelium. Unlike other viral enterotoxins that have been identified, astrovirus capsid induces diarrhea after oral administration, reproducing the natural route of infection and demonstrating that ingestion of intact noninfectious capsid protein may be sufficient to provoke acute diarrhea. Based on these data, we hypothesize that the astrovirus capsid acts like an enterotoxin and induces intestinal epithelial barrier dysfunction.

Crystal Structure of the Human Astrovirus Capsid Protein.

ABSTRACTHuman astrovirus (HAstV) is a leading cause of viral diarrhea in infants and young children worldwide. HAstV is a nonenveloped virus with a T=3 capsid and a positive-sense RNA genome. The capsid protein (CP) of HAstV is synthesized as a 90-kDa precursor (VP90) that can be divided into three linear domains: a conserved N-terminal domain, a hypervariable domain, and an acidic C-terminal domain. Maturation of HAstV requires proteolytic processing of the astrovirus CP both inside and outside the host cell, resulting in the removal of the C-terminal domain and the breakdown of the rest of the CP into three predominant protein species with molecular masses of ∼34, 27/29, and 25/26 kDa, respectively. We have now solved the crystal structure of VP9071–415 (amino acids [aa] 71 to 415 of VP90) of human astrovirus serotype 8 at a 2.15-Å resolution. VP9071–415 encompasses the conserved N-terminal domain of VP90 but lacks the hypervariable domain, which forms the capsid surface spikes. The structure of VP9071–415 is comprised of two domains: an S domain, which adopts the typical jelly-roll β-barrel fold, and a P1 domain, which forms a squashed β-barrel consisting of six antiparallel β-strands similar to what was observed in the hepatitis E virus (HEV) capsid structure. Fitting of the VP9071–415 structure into the cryo-electron microscopy (EM) maps of HAstV produced an atomic model for a continuous, T=3 icosahedral capsid shell. Our pseudoatomic model of the human HAstV capsid shell provides valuable insights into intermolecular interactions required for capsid assembly and trypsin-mediated proteolytic maturation needed for virus infectivity. Such information has potential applications in the development of a virus-like particle (VLP) vaccine as well as small-molecule drugs targeting astrovirus assembly/maturation.IMPORTANCE Human astrovirus (HAstV) is a leading cause of viral diarrhea in infants and young children worldwide. As a nonenveloped virus, HAstV exhibits an intriguing feature in that its maturation requires extensive proteolytic processing of the astrovirus capsid protein (CP) both inside and outside the host cell. Mature HAstV contains three predominant protein species, but the mechanism for acquired infectivity upon maturation is unclear. We have solved the crystal structure of VP9071–415 of human astrovirus serotype 8. VP9071–415 encompasses the conserved N-terminal domain of the viral CP. Fitting of the VP9071–415 structure into the cryo-EM maps of HAstV produced an atomic model for the T=3 icosahedral capsid. Our model of the HAstV capsid provides valuable insights into intermolecular interactions required for capsid assembly and trypsin-mediated proteolytic maturation. Such information has potential applications in the development of a VLP vaccine as well as small-molecule drugs targeting astrovirus assembly/maturation.

Human rotavirus infection. Strategies for the vaccinal prevention.

Rotavirus was first isolated in 1973 in Australia from children with diarrhea. Hundreds of thousands of children die annually in developing countries from this virus with the mortality peaks in the most impoverished among them. According to wHo, rotavirus infection claims about 440 thousands children lives each year, being third in the mortality rate after pneumonia and malaria. Rotavirus is widely spread throughout the world and by the age of five years almost every child encountered this pathogen at least once. Rotavirus has a high genetic and antigenic diversity. The most important for humans is the group A rotavirus, and the most common by far genotypes are G1P [8], G2P [4], G3P [8], G4P [8], G9P [8], and to a lesser extent G12P [8]. There are three gene constellations described in rotavirus designated Wa, Ds-1, and Au-1. It is believed that they originated from rotaviruses of pigs, cattle, dogs, and cats, respectively. Cases of rotavirus interspecies transmission from animal to humans were reported. The first vaccines against rotavirus infection were based on naturally attenuated virus of the animal origin. Their efficiency, especially in developing countries, was inadequate, but today China and India use vaccines based on animal rotaviruses. Using the method of gene reassortation with the cattle rotavirus WC3 as a backbone, pentavalent vaccine against most common human rotavirus serotypes was developed and now successfully used as RotaTeq. The ability of rotavirus to protect against heterologous isolates was taken into account in the development of other vaccine, Rotarix, created on the basis of rotavirus genotype G1P1A [8]. The efficacy of these vaccines in developing countries is significantly reduced (51%), the cost of a dose is high, and so the search for more effective, safe, and inexpensive vaccines against rotavirus continues around the world.

Chimpanzee adenovirus vector-based avian influenza vaccine completely protects mice against lethal challenge of H5N1

Highly pathogenic avian H5N1 viruses may give rise to the next influenza pandemic due to their reassortment and mutation of the genome. Vaccine against this virus is important for coping with its potential threat. Chimpanzee adenovirus (Ad) vectors are a novel type of vaccine vectors that share the advantages of human serotype Ad vectors but without being affected by pre-existing human neutralizing antibody to the vaccine vector. Based on a replication-deficient chimpanzee Ad vector, AdC7, we generated a novel H5N1 vaccine candidate AdC7-H5HA that expresses H5N1 Hemagglutinin(HA). When tested in mice, the vaccine significantly reduced the virus load and pathological lesions in the lung tissues, and conferred complete protection against lethal challenge by a homologous virus. Mechanistically, the AdC7-H5HA vaccine can induce both HA-specific humoral and cell-mediated immune responses in mice. Also, sera transfer experiments demonstrated that neutralizing antibodies alone could provide protection. In conclusion, our results show that chimpanzee Ad vector expressing influenza virus HA may represent a promising vaccine candidate for H5N1 viruses and other influenza virus subtypes.

Genetic and pathogenic difference between Streptococcus agalactiae serotype Ia fish and human isolates

BackgroundStreptococcus agalactiae (GBS) is a common pathogen to infect newborn, woman, the elderly, and immuno-compromised human and fish. 37 fish isolates and 554 human isolates of the GBS in 2007–2012 were investigated in serotypes, antibiotic susceptibility, genetic difference and pathogenicity to tilapia.ResultsPCR serotyping determined serotype Ia for all fish GBS isolates and only in 3.2 % (3–4.2 %) human isolates. For fish isolates, all consisted a plasmid less than 6 kb and belonged to ST7 type, which includes mainly pulsotypes I and Ia, with a difference in a deletion at the largest DNA fragment. These fish isolates were susceptible to all antimicrobials tested in 2007 and increased in non-susceptibility to penicillin, and resistance to clindamycin and ceftriaxone in 2011. Differing in pulsotype and lacking plasmid from fish isolates, human serotype Ia isolates were separated into eight pulsotypes II–IX. Main clone ST23 included pulsotypes II and IIa (50 %) and ST483 consisted of pulsotype III. Human serotype Ia isolates were all susceptible to ceftriaxone and penicillin and few were resistant to erythromycin, azithromycin, clindamycin, levofloxacin and moxifloxacine with the resistant rate of 20 % or less. Using tilapia to analyze the pathogenesis, fish isolates could cause more severe symptoms, including hemorrhage of the pectoral fin, hemorrhage of the gill, and viscous black and common scites, and mortality (>95 % for pulsotype I) than the human isolates (<30 %); however, the fish pulostype Ia isolate 912 with deletion caused less symptoms and the lowest mortality (<50 %) than pulsotype I isolates.ConclusionGenetic, pathogenic, and antimicrobial differences demonstrate diverse origin of human and fish serotype Ia isolates. The pulsotype Ia of fish serotype Ia isolates may be used as vaccine strains to prevent the GBS infection in fish.Electronic supplementary materialThe online version of this article (doi:10.1186/s12866-016-0794-4) contains supplementary material, which is available to authorized users.

T- and B-cell responses to multivalent prime-boost DNA and viral vectored vaccine combinations against hepatitis C virus in non-human primates.

Immune responses against multiple epitopes are required for the prevention of hepatitis C virus (HCV) infection, and the progression to phase I trials of candidates may be guided by comparative immunogenicity studies in non-human primates. Four vectors, DNA, SFV, human serotype 5 adenovirus (HuAd5) and Modified Vaccinia Ankara (MVA) poxvirus, all expressing hepatitis C virus Core, E1, E2 and NS3, were combined in three prime-boost regimen, and their ability to elicit immune responses against HCV antigens in rhesus macaques was explored and compared. All combinations induced specific T-cell immune responses, including high IFN-γ production. The group immunized with the SFV+MVA regimen elicited higher E2-specific responses as compared with the two other modalities, while animals receiving HuAd5 injections elicited lower IL-4 responses as compared with those receiving MVA. The IFN-γ responses to NS3 were remarkably similar between groups. Only the adenovirus induced envelope-specific antibody responses, but these failed to show neutralizing activity. Therefore, the two novel regimens failed to induce superior responses as compared with already existing HCV vaccine candidates. Differences were found in response to envelope proteins, but the relevance of these remain uncertain given the surprisingly poor correlation with immunogenicity data in chimpanzees, underlining the difficulty to predict efficacy from immunology studies.Supplementary informationThe online version of this article (doi:10.1038/gt.2016.55) contains supplementary material, which is available to authorized users.

Mechanism of human rhinovirus infections.

About 150 human rhinovirus serotypes are responsible for more than 50 % of recurrent upper respiratory infections. Despite having similar 3D structures, some bind members of the low-density lipoprotein receptor family, some ICAM-1, and some use CDHR3 for host cell infection. This is also reflected in the pathways exploited for cellular entry. We found that even rhinovirus serotypes binding the same receptor can travel along different endocytic pathways and release their RNA genome into the cytosol at different locations. How this may account for distinct immune responses elicited by various rhinoviruses and the observed symptoms of the common cold is briefly discussed.

257. Selection of Next Generation AAV Gene Therapy Vectors for Specific and Precise Gene Delivery

The ultimate goal in human gene therapy is the specific and exclusive modification of the desired target cells upon systemic vector administration. Especially vectors derived from adeno-associated virus (AAV) are among the most promising gene transfer systems for in vivo application and have received broad attention due to substantial clinical benefit. However, AAV specificity for a particular target cell or tissue has been hampered by the broad tropism of different AAV serotypes. Over the last several years, new approaches have been initiated to create and select for more effective and selective recombinant AAV vectors by genetically modifying the capsid protein. These methods include random and/or rationale amino acid substitutions, creation of chimeric capsid variant libraries and various selection screens, and/or peptide insertion. A different approach involves the incorporation of highly specific binding molecules (DARPins) into the AAV capsid. DARPins are derived from ankyrin-repeat proteins that have been developed as alternative to antibody-based scaffolds, which are selected by high-throughput screens from DARPin libraries. At present, the bottleneck remains the cumbersome selection of DARPin molecules and the low number of functional DARPin capsid chimeras, which are able to assemble into a functional vector and still bind their intended extracellular target. Thus after months of screening and analysis the majority of the pre-selected DARPins are found not to be suitable for AAV targeting approaches. Here we describe a novel reliable and faster selection approach. First we eliminate the cumbersome and often misleading prokaryotic selection steps. To do this, we incorporate an entire DARPin library into the AAV capsid as VP2-fusion protein, generating either replication deficient or competent AAV library systems allowing for direct screening on therapeutically relevant cell types in vitro or in vivo. Our studies show that replicating deficient and replicating AAV libraries can be generated with high diversity up to 5×107 and high functional titers retaining their infectivity. We have generated a DARPin-capsid library in AAV-DJ (serotype with diverse cellular tropism) and AAV-LK03 (human selective serotype) and after only two selection rounds, found up to 10% of selected DARPins sharing identical repeat motifs responsible for target receptor binding in the pancreas and the liver. We are currently making vectors from these chimeric capsids and will determine their target specificity both in vivo and in vitro. This new approach takes only six days per selection cycle, allowing discovery of novel DARPin molecules and selection for targeted vectors in substantially reduced time. This approach expands the potential diversity in creating and defining novel rAAV vectors with medically relevant transduction properties. Such viral vectors will not only open the door for an array of new approaches to treat acquired human diseases but also push the development of new AAV-based gene therapy vectors to the next level.

293. Discovery of an Essential Receptor for Adeno-Associated Virus Infection

Cellular entry of adeno-associated viruses (AAV) is poorly understood, despite the prominent use of AAV vectors in gene therapy for several monogenic diseases. Using an unbiased, haploid genetic screen, we identified critical players in AAV serotype 2 (AAV2) entry including members of distinct protein complexes involved in retrograde trafficking as well as genes involved in the biosynthesis of the AAV2 attachment factor, heparan sulphate. We focused on the single most significantly enriched gene of the screen, an uncharacterized type-I transmembrane protein. Based on the evidence below we renamed this gene AAV receptor (AAVR). We discovered AAVR as capable of rapidly endocytosing from the plasma membrane and trafficking to the trans-Golgi network, taking a similar path as AAV particles utilize. Genetic ablation of AAVR using CRISPR/Cas9 technology demonstrated a robust resistance to AAV2 infection in a wide range of mammalian cell types, which could be reversed upon AAVR complementation. This confirmed the essentiality of AAVR in AAV2 infection. Further investigation revealed that AAVR was also required for the infections of all tested human and simian-derived AAV serotypes including AAV1, 3B, 5, 6, 8 and 9. Deeper characterization of AAVR showed it to contain Ig-like domains, which are commonly found in many virus receptors including those for poliovirus and measles. We observed that these domains are capable of binding to AAV2 particles, and anti-AAVR antibodies efficiently block AAV2 infection. Moreover, the importance of AAVR for AAV infection in vivo was demonstrated by the strong resistance of AAVR−/− mice to AAV9 infection, recapitulating what we showed in vitro. Collectively, the data indicates that AAVR is a universal receptor involved in AAV infection. This has significant implications for the improvement of future AAV vector design.

Estimation of the potential impact of dengue vaccination on clinical outcomes in Brazil

Objective: The aim of the current analysis was to measure the public health impact of dengue vaccination in Brazil using a published transmission dynamics model. Methods: We adapted a mathematical model that represented the transmission dynamics of the four dengue fever serotypes in humans and in the mosquito. This compartmental model represents the known characteristics of dengue transmission dynamics: host-vector interactions, immunological interactions between all four dengue serotypes, age structure of the population, levels of specific transmission by age, seasonality of the disease, and growth of the human and vector population. Results: Our mathematical model showed a 22% (CI95%: 9-37) reduction of all cases of dengue fever for a smaller scenario (routine vaccination at 9 years old and catch-up campaign to 10 years of age) and 81% (CI95%: 67-89) in the largest scenario (routine vaccination at 9 years old and catch-up campaign to 40 years of age) over a 5-year period. For the 10-year impact, we estimated a 22% (CI95%: 12-39) reduction in the smaller scenario, and a 92% (CI95%: 80-95) reduction in the larger scenario. This reduction in the number of cases would lead to significant decrease in the number of hospitalizations. Up to 233,509 (CI95%: 148,534 - 331,849) and 739,378 (CI95%: 604,386 – 894,072) hospitalizations would be prevented over a 5-year and 10-year period, respectively, with the larger vaccination program. Conclusion: This analysis indicates that, within expected variations, a national dengue vaccination program in Brazil would lead to significant public health benefits by reducing dengue infections and hospitalizations.

Development of intranasal nanovehicles of itraconazole and their immunological activities for the therapy of rhinovirus infection

Itraconazole (ITZ)-loaded microemulsion (ME) systems for intranasal (IN) delivery were developed for the treatment of human rhinovirus serotype 1B (HRV1B) infection. ITZ was incorporated into the oil-in-water (o/w) ME formulation composed of benzyl alcohol (oil), Cremophor EL (surfactant), Solutol HS15 (cosurfactant), and water. The optimized composition of ME was determined by constructing pseudo-ternary phase diagram. ITZ ME formulation with about 150nm mean diameter and spherical shape was prepared and the solubility of ITZ in blank ME was markedly improved (up to 13.9mg/mL). The initial value of droplet size was maintained with four times dilution in the aqueous buffer and 72h incubation. Released amounts of drug from ME formulation were significantly enhanced compared to drug suspension group (p<0.05). Particularly, ITZ ME group displayed lower levels of inflammatory markers in the lung compared to ITZ suspension group after their IN administration in the HRV1B-infected mouse model (p<0.05). Developed ITZ ME formulation via IN route can be a promising candidate for the treatment of rhinovirus infection.