Circulating Neutralizing Antibodies Research Articles

P640 Role of vedolizumab trough levels in predicting clinical response in Inflammatory Bowel Disease: a multicentre prospective study

Abstract Background Medical treatment of Inflammatory Bowel Diseases (IBD) has developed aiming at a target-therapy using molecular-targeted drugs, such as vedolizumab (VDZ), recommended in moderate to severe forms of IBD refractory to conventional therapy. Considering the low rate of clinical remission after 12 months of treatment (about 30%) and its cost, it is important to identify predictors of clinical efficacy: therapeutic drug monitoring (TDM) based on measuring VDZ trough levels (TL) and circulating neutralizing antibodies could be useful in this setting. This prospective study aims at evaluating the association between VDZ TLs at week 24 and clinical and biochemical disease activity at the same timepoint and at week 52, in order to identify if these levels can predict 12-months clinical remission Methods The study has included patients who started VDZ treatment from 2016 to 2022 at two IBD centres (Turin and Pisa). Patients were monitored through clinical scores (HBI and PMS) and laboratory tests (haemoglobin, CRP, faecal calprotectin) at the beginning of treatment, at 6, at 12-months; TDM has been performed at month 6; response to the treatment and clinical remission have been defined according to IOIBD criteria after 6 and 12 months Results All of 40 patients included (16 CD, 24 UC) have undergone a follow-up at month 6, while only 35 have been to an annual evaluation. At the beginning of treatment the clinical disease activity was moderate (HBI mean 7.5; PMS mean 4.9), it became mild at the second timepoint (HBI mean 3.5; PMS mean 2.5) and was found to be mild or in remission after a year (HBI mean 3.6; PMS mean 1.5). The response rate (77%) and clinical remission rate (63%) at 6 and 12-months have been steady over time. TDM has found a mean of VDZ TLs of 79.3 μg/ml (SD 51.3) and no anti-VDZ antibodies. 12-months clinical remission has been tested through logistic regression in order to identify predictive factors: it was inversely proportional to PMS (OR 0.47; p 0.02) and abnormal CRP levels (OR 0.24; p 0.04) at 6-months and directly associated with haemoglobin concentration at 0 (OR 2.14; p 0.04) and 6-months (OR 1.86; p 0.02). As to TDM, VDZ TLs at the second timepoint were positive predictors of symptoms remission at the next timepoint (OR 1.02; p 0.03): higher concentrations at 43.1 μg/ml were associated with clinical remission (AUC 0.69; p 0.03) and were positively correlated with laboratory tests, such as haemoglobin (r 0.36; p 0.03) Conclusion Response to VDZ at the end of the year can be predicted after the first 6 months with clinical evaluation and laboratory tests, as well as TDM: lower PMS, higher haemoglobin, quiescent inflammatory biomarkers together with higher VDZ TLs allow to identify who will achieve clinical remission.

Immunogenicity of poxvirus-based vaccines against Nipah virus

Nipah virus (NiV), an emerging zoonotic pathogen in Southeast Asia, is transmitted from Pteropus species of fruit bats to a wide range of species, including humans, pigs, horses, dogs, and cats. NiV has killed millions of animals and caused highly fatal human outbreaks since no vaccine is commercially available. This study characterized the immunogenicity and safety of poxvirus-based Nipah vaccines that can be used in humans and species responsible for NiV transmission. Mice were vaccinated with modified vaccinia Ankara (MVA) and raccoon pox (RCN) viral vectors expressing the NiV fusion (F) and glycoprotein (G) proteins subcutaneously (SC) and intranasally (IN). Importantly, both vaccines did not induce significant weight loss or clinical signs of disease while generating high circulating neutralizing antibodies and lung-specific IgG and IgA responses. The MVA vaccine saw high phenotypic expression of effector and tissue resident memory CD8ɑ+ T cells in lungs and splenocytes along with the expression of central memory CD8ɑ+ T cells in lungs. The RCN vaccine generated effector memory (SC) and tissue resident (IN) CD8ɑ+ T cells in splenocytes and tissue resident (IN) CD8ɑ+ T cells in lung cells. These findings support MVA-FG and RCN-FG viral vectors as promising vaccine candidates to protect humans, domestic animals, and wildlife from fatal disease outcomes and to reduce the global threat of NiV.

Comparison of Two Diagnostic Assays for the Detection of Serum Neutralizing Antibody to Porcine Epidemic Diarrhea Virus

Lactogenic immunity is important for the protection of piglets against many pathogens including porcine epidemic diarrhea virus. Circulating neutralizing antibodies levels in sow sera may help determine if a detectable immune response could confer protection to piglets. Neutralizing antibodies can be detected through various diagnostic assays. This study evaluated the diagnostic characteristics of two neutralizing antibody assays for porcine epidemic diarrhea virus neutralizing antibodies in serum of challenged gilts. Four treatment groups, control, non-vaccinated, vaccinated prior to challenge, and vaccinated following challenge, were comprised of 20 gilts. Serum sample were collected from each gilt prior to and following challenge with porcine epidemic diarrhea virus. Samples were evaluated for the presence of neutralizing antibodies via a fluorescent focus neutralization assay and a high-throughput neutralization assay. Diagnostic sensitivity and specificity for the fluorescent focus neutralization and high-throughput neutralization assays for this study were optimized at a cutoff of a dilution of 80 and 80% fluorescent reduction respectively and demonstrated moderate agreement based off the kappa statistic. The focus fluorescent neutralization and high-throughput neutralization assays can be used to monitor the status of neutralizing antibodies within animals or a population of animals. The high-throughput assay has advantages over the focus fluorescent assay in that it has a higher specificity at the indicated cut-off and the nature of the results allows for more discrimination between individual results.

Timing and Immune Status after Cellular Therapies Predict Response to COVID-19 Vaccines

BACKGROUNDCellular therapies (allogeneic hematopoietic cell transplantation, allo-HCT, autologous hematopoietic cell transplantation, auto-HCT, and chimeric antigen receptor T cell therapy, CAR T) render patients severely immunocompromised for extended periods post-therapy. Emerging data suggest reduced immune responses to COVID-19 vaccines among patients with hematologic malignancies, but data for cellular therapy recipients are sparse. We therefore assessed immune responses to mRNA COVID-19 vaccines among patients who underwent cellular therapies at our center to identify predictors of response.PATIENT AND METHODSIn this observational prospective study, anti-SARS-CoV-2 spike IgG antibody titers and circulating neutralizing antibodies were measured at 1 and 3 months after the 1 st dose of vaccination. CD4, CD19, mitogen, and IgG levels from patient samples collected prior to initiation of vaccination in a subset of patients were used to assess immune recovery and association with response. A concurrent healthy donor (HD) cohort provided control response rates.RESULTSAllo-HCT (N=149), auto HCT (N=61), and CAR T (N=7) patients vaccinated between 12/22/2020- 2/28/2021 with mRNA vaccines and 69 HD participated in this study. At 3 months, 188 pts (87%) had a positive anti-SARS-CoV-2 spike IgG levels (median 5,379 AU/mL, IQR 451-15,750), and 139 (77%) had a positive neutralization Ab assay (median 93%, IQR 36-96%). All HD (100%) had a positive anti-SARS-CoV-2 spike IgG and a positive neutralization Ab assay with median levels of 8,011 AU/mL (IQR 4573-11,159) and 96% (IQR 78- 96%), respectively.Time from vaccination to cellular therapy was associated with response; 67% of patients vaccinated in the first 12 months post-cellular therapy (N=42) mounted a serologic response, compared with patients vaccinated between 12-24 (89%) (N=45), 24-36 (91%) (N=32) and >36 (93%) (N=98) months post-treatment, p= 0.001 (figure 1). Patients with immune parameters below the recommended threshold for vaccinations post-cellular therapies were also less likely to mount a response (figure 2): CD4+ T-cell count < 200 vs >200 cells/μL, 66% vs 87% (p=0.012); CD19+ B-cell count <50 vs >50 cells/μL; 33% vs 95% (p<0.001), phytohemagglutinin mitogen response <40% vs >40%, 42% vs 89% (p<0.001), and IgG <500 vs >500 mg/dl, 71% vs 91% (p=0.003). Patient age, gender, prior COVID-19 infection, treatment with IVIG, and type of mRNA COVID-19 vaccine were not associated with the likelihood of serologic response.CONCLUSIONThis largest cohort to date, demonstrates that COVID-19 vaccine responses of cellular therapy recipients are reduced compared to healthy control and response varies based on time interval from cellular therapy and immune function at the time of vaccination, underscoring the importance of monitoring immune status parameters, as well as qualitative measures (neutralizing Ab) of vaccine response, in informing clinical decisions, including the indication for booster vaccines. [Display omitted] DisclosuresPolitikos: Merck: Research Funding; ExcellThera, Inc: Other: Member of DSMB - Uncompensated. Vardhana: Immunai: Membership on an entity's Board of Directors or advisory committees. Perales: Equilium: Honoraria; Cidara: Honoraria; Sellas Life Sciences: Honoraria; Miltenyi Biotec: Honoraria, Other; Celgene: Honoraria; MorphoSys: Honoraria; Takeda: Honoraria; Incyte: Honoraria, Other; Karyopharm: Honoraria; Kite/Gilead: Honoraria, Other; Merck: Honoraria; NexImmune: Honoraria; Novartis: Honoraria, Other; Medigene: Honoraria; Omeros: Honoraria; Servier: Honoraria; Bristol-Myers Squibb: Honoraria; Nektar Therapeutics: Honoraria, Other. Shah: Amgen: Research Funding; Janssen Pharmaceutica: Research Funding.

Humoral immune response following prime and boost BNT162b2 vaccination in people living with HIV on antiretroviral therapy.

ObjectivesPeople living with HIV (PLWH) with low CD4 T‐cell counts may be at a higher risk for severe coronavirus disease 2019 (COVID‐19) outcomes and in need of efficient vaccination. The World Health Organization (WHO) now recommends prioritizing PLHIV for COVID‐19 vaccination. Data on immune responses after messenger RNA (mRNA) vaccination in PLHIV in relation to CD4 counts are scarce. We aimed at assessing the humoral immune response in PLHIV after mRNA vaccination against COVID‐19.MethodsWe examined a cohort of PLHIV after prime (n = 88) and boost (n = 52) vaccination with BNT162b2. We assessed levels of anti‐severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) spike (S) protein‐specific immunoglobulin G (IgG)/IgA and circulating neutralizing antibodies in plasma and correlated results to the cellular immune status. BNT162b2‐vaccinated health care workers served as controls.ResultsAll PLWH had a viral load of ≤ 200 HIV‐1 RNA copies/mL and 96.5% had a viral load of < 50 copies/mL. Anti‐S IgG and neutralizing antibody responses after BNT162b2 priming were significantly lower in PLHIV having a CD4:CD8 T‐cell ratio of < 0.5. However, we observed robust humoral immunity in the majority of PLWH receiving antiretroviral therapy (ART) irrespective of CD4 T‐cell nadir, current CD4 count or CD4:CD8 ratio after full BNT162b2 vaccination. Nevertheless, HIV‐negative controls produced significantly higher mean anti‐S IgG concentrations with less variability.ConclusionsThe majority of PLWH mounted robust responses after complete BNT162b2 vaccination but overall amounts of antibodies directed against the SARS‐CoV‐2 receptor‐binding domain were variable. The impact on clinical efficacy remains unclear.

COVID-19 convalescent plasma cohort study: Evaluation of the association between both donor and recipient neutralizing antibody titers and patient outcomes.

BackgroundCurrent evidence regarding COVID‐19 convalescent plasma (CCP) transfusion practices is limited and heterogeneous. We aimed to determine the impact of the use of CCP transfusion in patients with previous circulating neutralizing antibodies (nAbs) in COVID‐19.MethodsProspective cohort including 102 patients with COVID‐19 transfused with ABO compatible CCP on days 0–2 after enrollment. Clinical status of patients was assessed using the adapted World Health Organization (WHO) ordinal scale on days 0, 5, and 14. The nAbs titration was performed using the cytopathic effect‐based virus neutralization test with SARS‐CoV‐2 (GenBank MT126808.1). The primary outcome was clinical improvement on day 14, defined as a reduction of at least two points on the adapted WHO ordinal scale. Secondary outcomes were the number of intensive care unit (ICU)‐free days and the number of invasive mechanical ventilation‐free days.ResultsBoth nAbs of CCP units transfused (p < 0.001) and nAbs of patients before CCP transfusions (p = 0.028) were associated with clinical improvements by day 14. No significant associations between nAbs of patients or CCP units transfused were observed in the number of ICU or mechanical ventilation‐free days. Administration of CCP units after 10 days of symptom onset resulted in a decrease in ICU‐free days (p < 0.001) and mechanical ventilation‐free days (p < 0.001).ConclusionTransfusion of high titer nAbs CCP units may be a determinant in clinical strategies against COVID‐19. We consider these data as useful parameters to guide future CCP transfusion practices.

Revisiting rabies virus neutralizing antibodies through infecting BALB/c mice with live rabies virus

This study investigates the production of rabies virus (RABV) neutralizing antibody after virus infection through a mouse model. The BALB/c mice from different age groups (three, five, seven week old) were intramuscularly inoculated with live rabies virus (TX coyote 323R). Without pre-exposure or post-exposure prophylaxis (PEP), we found there is a decreased fatality with increased age of animals, the mortalities are 60%, 50%, and 30%, respectively. Interestingly, through assay of rapid fluorescent focus inhibition test (RFFIT), direct fluorescent antibody (DFA) and quantitative Polymerase Chain Reaction (qPCR), the results showed that all the animals that succumbed to rabies challenge, except one, developed circulating neutralizing antibodies, and all the healthy animals, except two, did not generate virus neutralizing antibodies (VNA). Our animal study suggests that the induction of VNA was an indicator of infection progression in the central nervous system (CNS) and speculate that RABV neutralizing antibodies did not cross the blood-brain barrier of the CNS for those diseased animals. We hypothesize that early release of viral antigens from damaged nerve tissue might potentially be a benefit for survivors, and we also discuss several other aspects of the interaction of RABV and its neutralizing antibodies.

Optimized AAV rh.10 Vectors That Partially Evade Neutralizing Antibodies during Hepatic Gene Transfer.

Of the 12 common serotypes used for gene delivery applications, Adeno-associated virus (AAV)rh.10 serotype has shown sustained hepatic transduction and has the lowest seropositivity in humans. We have evaluated if further modifications to AAVrh.10 at its phosphodegron like regions or predicted immunogenic epitopes could improve its hepatic gene transfer and immune evasion potential. Mutant AAVrh.10 vectors were generated by site directed mutagenesis of the predicted targets. These mutant vectors were first tested for their transduction efficiency in HeLa and HEK293T cells. The optimal vector was further evaluated for their cellular uptake, entry, and intracellular trafficking by quantitative PCR and time-lapse confocal microscopy. To evaluate their potential during hepatic gene therapy, C57BL/6 mice were administered with wild-type or optimal mutant AAVrh.10 and the luciferase transgene expression was documented by serial bioluminescence imaging at 14, 30, 45, and 72 days post-gene transfer. Their hepatic transduction was further verified by a quantitative PCR analysis of AAV copy number in the liver tissue. The optimal AAVrh.10 vector was further evaluated for their immune escape potential, in animals pre-immunized with human intravenous immunoglobulin. Our results demonstrate that a modified AAVrh.10 S671A vector had enhanced cellular entry (3.6 fold), migrate rapidly to the perinuclear region (1 vs. >2 h for wild type vectors) in vitro, which further translates to modest increase in hepatic gene transfer efficiency in vivo. More importantly, the mutant AAVrh.10 vector was able to partially evade neutralizing antibodies (~27–64 fold) in pre-immunized animals. The development of an AAV vector system that can escape the circulating neutralizing antibodies in the host will substantially widen the scope of gene therapy applications in humans.

Occurrence of Escape Favoring Mutations in The Targets of 2F5 and 4E10 Antibodies of HIVgp41 : A Metadata Analysis

Objective The preventive vaccine for HIV has been an unachieved goal and the increasing prevalence burdens the field of medicine for the last three decades. The challenge is due to the substantial overall sequence variability that is found across the genome of HIV- 1. Here a retrospection has been done to investigate the degree of conservation in the targets of the 2F5 and 4E10 neutralizing antibodies. The 6 and 7 amino acid long epitopes of the neutralizing antibodies are embedded in the MPER (Membrane Proximal External Region) of the HIV-1 glycoprotein 41. Methods HIV-1 gp 41 nucleotide sequences reported from different parts of the world were collected as fasta sequences on a random basis. The sequences were aligned and investigated for mutations found in the epitopes targeted by the 2F5 and 4E10 neutralizing antibodies. Results Substitutions and deletions almost in all the residues were found. The mutations that were found in each of the residue can critically affect the affinity of the neutralizing antibodies. The occurrence of these mutations in the critical residues in combination with the neighboring residues may offer a survival advantage for the virus to escape from these circulating neutralizing antibodies. This invariant nature which is substantiated earlier has been diverging over the years. Conclusion The mutations can crucially alter the dynamicity and the reactivity of the 2F5 and 4E10 antibodies directed to the putative vaccine targets. This confers the fact that these targets may be less appropriate a candidate for a vaccine. The infidel reverse transcriptase that copies these ‘selfish’ survivable mutants is characteristically favoring the virus to make progeny which can escape the surveillance by these neutralizing antibodies. Hence, due to the observed trend in the lack of conservation, it becomes necessary to search for better candidates for a stronger immune response that promises success in vaccine strategies.

Airway structural cells regulate TLR5-mediated mucosal adjuvant activity.

Antigen-presenting cell (APC) activation is enhanced by vaccine adjuvants. Most vaccines are based on the assumption that adjuvant activity of Toll-like receptor (TLR) agonists depends on direct, functional activation of APCs. Here, we sought to establish whether TLR stimulation in non-hematopoietic cells contributes to flagellin's mucosal adjuvant activity. Nasal administration of flagellin enhanced T-cell-mediated immunity, and systemic and secretory antibody responses to coadministered antigens in a TLR5-dependent manner. Mucosal adjuvant activity was not affected by either abrogation of TLR5 signaling in hematopoietic cells or the presence of flagellin-specific, circulating neutralizing antibodies. We found that flagellin is rapidly degraded in conducting airways, does not translocate into lung parenchyma and stimulates an early immune response, suggesting that TLR5 signaling is regionalized. The flagellin-specific early response of lung was regulated by radioresistant cells expressing TLR5 (particularly the airway epithelial cells). Flagellin stimulated the epithelial production of a small set of mediators that included the chemokine CCL20, which is known to promote APC recruitment in mucosal tissues. Our data suggest that (i) the adjuvant activity of TLR agonists in mucosal vaccination may require TLR stimulation of structural cells and (ii) harnessing the effect of adjuvants on epithelial cells can improve mucosal vaccines.

Superior protection conferred by inactivated whole virus vaccine over subunit and DNA vaccines against salmonid alphavirus infection in Atlantic salmon (Salmo salar L.)

Salmonid alphavirus 3 (SAV-3) is an emerging pathogen in Norwegian salmon farming and causes severe annual losses. We studied the immunogenicity and protective ability of subunit and DNA vaccines based on E1 and E2 spike proteins of salmonid alphavirus subtype 3 (SAV-3), and compared these to an experimental inactivated, whole virus (IWV) vaccine in Atlantic salmon. The antigens were delivered as water-in-oil emulsions for the subunit and inactivated vaccines and non-formulated for the DNA vaccines. The IWV and the E2 subunit prime–boost groups had circulating neutralizing antibodies at challenge, correlating with high protection against lethal challenge and 3-log10 reduction of virus titer in heart for the IWV group. Prime–boost with E1 subunit vaccine also conferred significant protection against mortality, but did not correlate with neutralizing antibody levels. Protection against pathology in internal organs was only seen for the IWV group. Prime–boost with E1 and E2 DNA vaccines showed marginal protection in terms of reduction of viral replication in target organs and protection against mortality was not different from controls. The IWV group showed significant upregulation of IFNγ and IL2 mRNA expression at 4 weeks post challenge possibly indicating that other mechanisms in addition to antibody responses play a role in mediating protection against infection. This is the first report comparing the immunogenicity and protection against mortality for IWV vaccines and spike protein subunit and DNA vaccines against salmonid alphavirus infection in Atlantic salmon. The IWV vaccine has superior immunogenicity over sub-unit and DNA vaccines.

The influence of epileptic neuropathology and prior peripheral immunity on CNS transduction by rAAV2 and rAAV5

Adeno-associated virus (AAV) provides a promising platform for clinical treatment of neurological disorders owing to its established efficacy and lack of apparent pathogenicity. To use viral vectors in treating neurological disease, however, transduction must occur under neuropathological conditions. Previous studies in rodents have shown that AAV5 more efficiently transduces cells in the hippocampus and piriform cortex than AAV2. Using the kainic acid (KA) model of temporal lobe epilepsy and AAV2 and 5 carrying a hybrid chicken β-actin promoter driving green fluorescent protein (GFP), we found that limbic seizure activity caused substantial neuropathology and resulted in a significant reduction in subsequent AAV5 transduction. Nonetheless, this reduced transduction still was greater than AAV2 transduction in control rats. Although KA seizures compromise blood-brain barrier function, potentially increasing exposure of target tissue to circulating neutralizing antibodies, we observed no interaction between KA seizure-induced damage and immunization status on AAV transduction. Finally, while we confirmed the near total neuronal-specific transgene expression for both serotypes in control rats, AAV5-GFP expression was increasingly localized to astrocytes in seizure-damaged areas. Thus, the pathological milieu of the injured brain can reduce transduction efficacy and alter viral tropism- both relevant concerns when considering viral vector gene therapy for neurological disorders.

Safety and efficacy of novel dermal and epidermal microneedle delivery systems for rabies vaccination in healthy adults

In the present pilot study, intradermal ID delivery systems with a BD microneedle from 1 to 3 mm in length, and epidermal delivery (BD skin abrader) through abraded skin surface relative to standard intramuscular injection were evaluated. Circulating neutralizing antibodies were measured against the rabies virus after the Vero cells rabies vaccine was administered at D0, D7, D21 and D49. This clinical evaluation in 66 healthy volunteers shows that ID delivery using BD microneedle technology of 1/4 the IM antigen dose is safe, efficient and reliable, resulting in a protective seroconversion rate. In contrast, the epidermal delivery route did not produce an immune response against the rabies vaccine.

Prolonged Susceptibility to Antibody-mediated Neutralization for Adeno-associated Vectors Targeted to the Liver

Adeno-associated virus (AAV) vectors demonstrate highly efficient gene transfer to hepatocytes in vivo. One of the remaining obstacles to the treatment of hemophilia B patients with AAV vectors is the sensitivity of these vectors to antibody-mediated neutralization following systemic delivery. Testing and implementation of strategies to circumvent pre-existing antibodies requires knowledge of the clearance kinetics of AAV from circulation. In this study, AAV clearance kinetics were established for serotypes 2 and 8 in cell culture and in mice. Administration of pooled neutralizing serum subsequent to administration of the vector was used to define the time period in which the vector is susceptible to antibody-mediated neutralization. These experiments defined the in vivo clearance rates for both AAV2 and AAV8 vectors to be between 2 and 4 hours. In mice, portal vein and tail vein administration of each vector was tested with similar results. Cell culture studies in W162 cells established that cellular attachment and internalization both contribute to the clearance kinetics of AAV vectors. These studies characterize the in vivo clearance rates of AAV vectors for the first time and guide the development of future strategies for the avoidance of antibody-mediated AAV vector neutralization.

785. A Novel Adeno Associated Virus Vector That Efficiently Transduces Airway Epithelium In Vivo and Can Be Re-Administered

Top of pageAbstract Airway|[ndash]|directed gene transfer has emerged as a promising approach for the treatment of cystic fibrosis and |[alpha]|-1-antitrypsin deficiency (AAT). It will be important to re-administer the vector for treatment of these chronic disorders. We have shown previously that AAV2/9 can efficiently target and transduce the murine upper and lower airways in vivo. As such we studied the performance of AAV2/9 in the setting of pre-existing immunity to homologous vector. C57Bl/6 mice were inoculated intratracheally with a single dose (1E+11 genome copies) of either AAV2/9 or AAV2/5 expressing AAT. We found that AAV2/9-mediated AAT expression was relatively stable for the duration of the experiment (9 months) and at the peak of gene expression was 60-fold higher than that of AAV2/5. Within two weeks of vector administration, high levels (>1:2560) of serum|[ndash]|circulating neutralizing antibodies (NAB) were observed in both AAV2/9 and AAV2/5 treated mice. Mice were divided into groups and designated groups were subjected to a second instillation of the same vector serotype expressing a different transgene (nLacZ) at specific time points after delivery of the AAV2/9- or AAV2/5|[ndash]| AAT vectors. Mice were sacrificed 21 days following the AAV|[ndash]| nLacZ vector instillation and the lung and nose processed for nLacZ expression. The serum NAB titers in the AAV2/9 treatment group remained high (average of 1:2560) up to day 112 and decreased to |[sim]|1:640 by day 217. Interestingly, we found that AAV2/9 was able to circumvent the AAV2/9-specific NABs, resulting in nLacZ gene transfer levels in nasal and lung airways similar to that of age-matched naive mice at all time points. In AAV2/5-treated mice, no transduced cells were seen in either the nasal or lung airways over a broad range of serum|[ndash]|circulating NAB titers (1:56000 to 1:1280). Some level of gene transfer was achieved with AAV2/5-nLacZ when administered 196 days after the AAT vector which was when serum NAB declined to 1:320|[ndash]|1:160. To explain why AAV2/9 can efficiently transduce the lung airway epithelium in the presence of high titer serum|[ndash]| circulating NAB, the bronchoalveolar lavage fluid (BALF) isolated from mice at the first time point (day 49) and at the end of the experiment (day 217) was analyzed for AAV-specific NABs. We did not observe any NAB titer, IgG or IgA responses to AAV2/9 vector. In contrast, when BALF from AAV2/5 treated mice was analyzed, NAB titers ranged from 1:160 to 1:1280, vector IgG responses from 1:100 to 1:12800 and vector IgA responses from 1:100|[ndash]|1:1600. Our results suggest that AAV2/9 is an excellent candidate for clinical applications that require long term transgene expression. We show that AAV2/9 can be re-administered to lung with out diminution of gene transfer efficiency while AAV2/5 can not. This finding further supports the development of AAV2/9 for use in clinical applications for chronic diseases in which vector re-administration will be necessary.

116. The Proposed AAV Serotypes 10 and 11 Serologically Cross-React with AAV8 and AAV4

Introduction: Vectors based on a variety of AAV isolates are currently being explored for application in gene therapy. This pursuit is driven by the fact that relatively subtle structural differences can dramatically affect vector biology in several ways relevant to gene therapy such as efficiency of gene transfer, tropism and interactions with the immune system of the host. Particularly, circulating neutralizing antibodies can blunt a successful gene therapy. Serological cross-reactivity between AAV isolates is not only used as a reflective measure of neutralization in vivo but also as a way of classification of primate AAVs. Recently AAV10 and AAV11 were proposed by Mori et al. (2004) as novel serotypes. AAV10 and AAV11 were found to be structurally closest to the previously described serotypes AAV8 and AAV4, respectively. Their study showed that the isolates from cynomolgus macaque were not cross-neutralized by polyclonal mouse antiserum generated against purified VP2 protein from AAV2, AAV4 or AAV8.

Determination of Sheep Pox Circulating Neutralizing Antibodies in Immunized Sheep and Goats in the North Region of the Kingdom of Saudi Arabia

Rashwan, S. M., Al Khalaf, H. N., Al Hammad, M. I., Abdel-Baky, M. H. and Azab, A. H. 2001. Determination of sheep pox circulating neutralizing antibodies in immunized sheep and goats in the North Region of the Kingdom of Saudi Arabia. J. Appl. Anim. Res., 19: 233–235. As a first field trial to evaluate the sheep pox vaccine efficacy under farm conditions, sheep pox virus-neutralizing antibodies were determined in vaccinated sheep and goats of different breeds, ages and sexes in one of the biggest mutton farms in the north region of the Kingdom. The results exhibited 100% post-vaccinal serum neutralizing antibodies with a titre range of 1.2 to 2.1 log 10 in sheep and 1.2 to 1.8 log 10 in goats.

Recombinant staphylokinase variants with altered immunoreactivity. IV: Identification of variants with reduced antibody induction but intact potency.

The thrombolytic potency and antibody induction of selected variants of recombinant staphylokinase (SakSTAR), including SakSTAR(K74) with Lys74, SakSTAR(E75) with Glu75-, SakSTAR(EER) with Glu38, Glu75, and Arg77, and SakSTAR(K74ER) with Lys74, Glu75, and Arg77 replaced by Ala, were studied. In rabbits, SakSTAR(74) and SakSTAR(EER) elicited significantly less circulating neutralizing activity than SakSTAR and SakSTAR(E75) (P = .005 and P = .0002 versus SakSTAR, respectively). In baboons, SakSTAR(K74) induced significantly fewer antibodies than wild-type SakSTAR (P < .05). Intra-arterial administration in patients with peripheral arterial occlusion of SakSTAR(K74) (n = 11) or SakSTAR(K74ER) (n = 6) induced significantly fewer circulating neutralizing antibodies [median values and interquartile ranges, 20 (3.8 to 26) and 2.4 (0.2 to 59) micrograms compound neutralized/mL plasma after 3 to 4 weeks (P = .01 and P = .035 versus SakSTAR, respectively)] than SakSTAR (n = 9) [median value and interquartile range, 93 (24 to 110) micrograms compound neutralized/mL plasma]. Overt neutralizing antibody induction (> 10 micrograms compound neutralized/mL plasma) occurred in all 9 patients given wild-type SakSTAR, in 6 of the 11 SakSTAR(K74ER) patients (P = .038 versus SakSTAR), and in 2 of the 6 SakSTAR(K74ER) patients (P = .011 versus SakSTAR). SakSTAR(K74), a variant of recombinant staphylokinase with a single substitution of Lys74 with Ala, and SakSTAR(K74), with Lys74, Glu75, and Arg77 substituted with Ala, have intact thrombolytic potencies but induce significantly less antibody formation in patients.

Herpesviruses in tortoises: investigations into virus isolation and the treatment of viral stomatitis in Testudo hermanni and T. graeca.

Various studies were done during a spontaneous outbreak of stomatitis-rhinitis-complex (mouth rot) in a collection of Mediterranean land tortoises (21 Testudo hermanni, Hermann's tortoises, and three Testudo graeca, spur-thighed tortoises) in southern Germany. These studies were intended to help diagnose the causative agent, establish a possible diagnostic method in vivo and provide information on the efficacy of aciclovir and ganciclovir against chelonian herpesviruses. Thirteen T. hermanni and no T. graeca died within a period of 6 weeks following the introduction of one apparently healthy T. graeca. Two of the dead Testudo hermanni were submitted for post-mortem examination. In addition, blood samples from 11 of the 12 tortoises still surviving at the beginning of this study were cultured for virus content and for the presence of neutralizing antibodies to chelonian herpesviruses and swabs from conjunctiva, pharynx and cloaca were cultured for the presence of viruses. Herpesviruses were isolated from tissues of the two dead Testudo hermanni (tongue, intestine, trachea, lung, spleen, heart and brain). Peripheral leukocytes from one of 11 blood samples were positive for herpesvirus isolation, indicating viremia in at least one animal. Nine of 11 pharyngeal swabs but none of the conjunctival and cloacal swabs yielded herpesviruses. Circulating neutralizing antibodies were present in two of two tested T. graeca, but absent in all of the nine samples from T. hermanni. Aciclovir and ganciclovir were effective when tested in vitro against one of the herpesvirus isolates.

Infection of pregnant guinea pigs with attenuated Junin virus strains.

The effect of the attenuated XJC13 and XJ0 strains of Junin virus (JV) was studied in guinea pigs infected before and during pregnancy. The 58% mortality rate in animals infected during gestation and the 16.7% mortality rate in chronically infected animals were attributed to a viral effect. An abortion rate of 33% occurred in animals infected before the 7th week of gestation. Regardless of the time of infection, JV was isolated from central nervous system tissue, placentas, and fetuses of animals killed just before parturition, even when circulating neutralizing antibodies were present. Results confirmed that transplacental infection is a regular event and showed that guinea pigs are more susceptible to attenuated JV strains during pregnancy, most probably due to immunosuppression, hormonal changes, or both.