Acinetobacter baumannii outer membrane protein A induces HeLa cell autophagy via MAPK/JNK signaling pathway.

Autophagy is an evolutionary conserved self-balancing process that plays an important role in maintaining cellular homeostasis via the clearance of damaged organelles and misfolded proteins. Infection-triggered autophagy specifically inhibits the invasion of intracellular bacterial replication and hence protects the cells from microbial infections. It has been reported that Acinetobacter baumannii trigger cell autophagy. However, the role of its virulence protein OmpA remains unclear. Therefore, this study aimed to explore the effects of Acinetobacter baumannii OmpA on cell autophagy and its underlying molecular mechanisms. The results showed that OmpA induced autophagy in HeLa and RAW264.7 cells, increased LC3BII expression, and hindered p62 degradation. Moreover, OmpA triggered incomplete autophagy by interfering the fusion of autophagosomes with lysosomes. Besides, OmpA activated MAPK/JNK signaling pathway and enhanced the phosphorylation levels of JNK, p38, and ERK, c-Jun. Inhibition of JNK signaling pathway suppressed OmpA-induced autophagy in HeLa cells. Ab wild-type strains carrying OmpA triggered incomplete autophagy and resulted in a large number of IL-1ß production. Ab-△OmpA strain (OmpA gene mutation) restored autophagic flux and reduced the accumulation of p62 and the release of IL-1ß in HeLa cells. Rapamycin activated autophagy to inhibit OmpA-induced IL-1ß secretion and protect HeLa cells from inflammatory damage. Collectively, these results suggest that OmpA can induce autophagy in HeLa cells through MAPK/JNK signaling pathway. Pre-treatment with Rapamycin activates autophagy and protects against cell death.

Saffold virus type 3 (SAFV-3) persists in HeLa cells.

Saffold virus (SAFV) was identified as a human cardiovirus in 2007. Although several epidemiological studies have been reported, they have failed to provide a clear picture of the relationship between SAFV and human diseases. SAFV genotype 3 has been isolated from the cerebrospinal fluid specimen of patient with aseptic meningitis. This finding is of interest since Theiler's murine encephalomyelitis virus (TMEV), which is the closely related virus, is known to cause a multiple sclerosis-like syndrome in mice. TMEV persistently infects in mouse macrophage cells in vivo and in vitro, and the viral persistence is essential in TMEV-induced demyelinating disease. The precise mechanism(s) of SAFV infection still remain unclear. In order to clarify the SAFV pathogenicity, in the present study, we studied the possibilities of the in vitro persistent infection of SAFV. The two distinct phenotypes of HeLa cells, HeLa-N and HeLa-R, were identified. In these cells, the type of SAFV-3 infection was clearly different. HeLa-N cells were lyticly infected with SAFV-3 and the host suitable for the efficient growth. On the other hand, HeLa-R cells were persistently infected with SAFV-3. In addition, the SAFV persistence in HeLa-R cells is independent of type I IFN response of host cells although the TMEV persistence in mouse macrophage cells depends on the response. Furthermore, it was suggested that SAFV persistence may be influenced by the expression of receptor(s) for SAFV infection on the host cells. The present findings on SAFV persistence will provide the important information to encourage the research of SAFV pathogenicity.

Preparation method of lamprey antisera and activity assay.

Lampreys, the surviving representative of jawless vertebrates, have been a focal point in the search for the evolutionary origin of adaptive immunity. They have independently evolved the variable lymphocyte receptor (VLR)-based adaptive immune system that protects themselves from infection by a variable of microorganisms. The standard immunization schedule for Japanese lamprey (Lampetra japonica) was established to prepare antisera by injection of Escherichia coli, Bacillus proteus, Staphylococcus aureus, Mycobacterium smegmatis, RRBCs, SRBCs, NB4 cells and Hela cells. In this study, we demonstrated the activities of lamprey antisera, which might be helpful to research the collaboration between VLR-based adaptive immune system and complement system in jawless vertebrates.

Increased concentration of sialidases by HeLa cells might influence the cytotoxic ability of NK cells.

OBJECTIVE: Cancer cells reportedly have the ability to escape from the immune system, mainly from natural killer (NK) cells. Although the real mechanisms are complicated, some inhibitors that are secreted from the cancer cells might play an important role. This study's aim was to investigate the potential mediator released by cancer cells (HeLa) that contributes to the decreased cytotoxicity of NK cells. METHODS AND MATERIALS: An NK-HeLa coculture system was used to test the hypothesis that the presence of the potential mediator from cancer cells contributes to the decreased cytotoxicity of NK cells. RESULTS: After coculturing with HeLa cancer cells, the cytotoxicity of NK cells was decreased. When the coculture medium and culture medium containing commercialized sialidase were used to culture NK cells, the cytotoxicity of the NK cells was also inhibited. However, cytotoxicity was partially restored by a sialidase inhibitor (DANA). Western blot analysis of the HeLa cells after coculturing with NK cells demonstrated increased Neu2 and Neu3 expression in HeLa cells. CONCLUSIONS: The finding that Neu2 and Neu3 expression in cancer cells might be involved in the impaired function of NK cells, which could be restored by a sialidase inhibitor, provides a new concept that could be applied to the management of cancer.

Altered ganglioside GD3 in HeLa cells might influence the cytotoxic abilities of NK cells.

OBJECTIVE: Previously, we found that altered sialidases in HeLa cells in a natural killer-HeLa (NK-HeLa) coculture system contributed to the decreased cytotoxic ability of NK cells. However, changes that occur in the glycosylation of the HeLa cells in the NK-HeLa coculture system remain unknown. MATERIALS AND METHODS: An NK-HeLa coculture system was used to examine the changes that occur in the gangliosides of HeLa cells. RESULTS: GD3 expression in HeLa cells was significantly increased in the NK-HeLa coculture system. Exogenous ganglioside GD3 decreased the cytotoxic ability of the NK cells, which could be restored by the addition of the anti-GD3 antibody. Coadministration of GD3 and sialidase further decreased the cytotoxic ability of the NK cells, which could be partially restored by the addition of a sialidase inhibitor (DANA). GD3 expression in HeLa cells also decreased following DANA treatment. CONCLUSIONS: This study suggests that interactions between ganglioside GD3 and sialidases in HeLa cells influence the cytotoxic ability of NK cells.

Entrapment of intracytosolic bacteria by septin cage-like structures.

Actin-based motility is used by various pathogens for dissemination within and between cells. Yet host factors restricting this process have not been identified. Septins are GTP-binding proteins that assemble as filaments and are essential for cell division. However, their role during interphase has remained elusive. Here, we report that septin assemblies are recruited to different bacteria that polymerize actin. We observed that intracytosolic Shigella either become compartmentalized in septin cage-like structures or form actin tails. Inactivation of septin caging increases the number of Shigella with actin tails and enhances cell-to-cell spread. TNF-α, a host cytokine produced upon Shigella infection, stimulates septin caging and restricts actin tail formation and cell-to-cell spread. Finally, we show that septin cages entrap bacteria targeted to autophagy. Together, these results reveal an unsuspected mechanism of host defense that restricts dissemination of invasive pathogens.

Cytokine profiles of HeLa and human diploid cells induced by different fractions of Vibrio parahaemolyticus cultures exposed to stress conditions.

Vibrio (V.) parahaemolyticus is an aquatic halophilic bacteria which produces gastroenteritis and in rare cases septicaemia after the consumption of raw or under-cooked contaminated seafood.The severity of diarrheal illness caused by this bacterium is closely related to the presence of two types of hemolysins (the thermostable direct hemolysin-TDH and TDH related hemolysin-TRH) and also of type III secretion system (TTSS) proteins. The TTSS type 1 induces a wide array of effects on infected HeLa cells such as autophagy, oncosis, cell rounding and lysis. Previous studies have shown that heat shock proteins have the ability to stimulate the production of interleukins in different cellular cultures. In our studies we have stimulated two cellular lines (HeLa and human diploid cells) with different V. parahaemolyticus culture fractions in order to observe the effect on cytokines production. Thus, the purpose of this study was to analyze the expression of IL-1, IL-2, IL-4, IL-6, IL-10 and TNF-alpha induced by the cell treatment with total cellular lysate, periplasmic fractions and culture supernatants extracted from V. parahaemolyticus exposed to normal and also to stress conditions. The ELISA assay of the cytokine profile of the HeLa and HDC cell lines stimulated with different bacterial fractions revealed that in the V. parahemolyticus cultures submitted to osmotic and heat shock stress are accumulating factors (probably heat shock proteins) which are exhibiting immunomodulatory activity, responsible for the induction of a pro-inflammatory response associated with increased levels of IL-6 and TNF-alpha expression, however balanced by the stimulation of the anti-inflammatory cytokine IL-4 synthesis.

Intrabody expression in eukaryotic cells.

We describe procedures for intracellular expression of scFv in eukaryotic cells. Starting from a scFv gene cloned in a phage-display vector, we describe the cloning step into a mammalian expression vector, the transient transfection of a HeLa cell line, and the monitoring of intrabody expression by immunofluorescence staining and FACS analysis.

Activation of CXCR4 triggers ubiquitination and down-regulation of major histocompatibility complex class I (MHC-I) on epithelioid carcinoma HeLa cells.

Many cancer cells display down-regulated major histocompatibility complex (MHC) class I antigen (MHC-I), which seems to enable them to evade immune surveillance, whereas the underlying mechanisms remain incompletely understood. Here, we demonstrate that ligand (CXCL12) stimulation of CXCR4, a major chemokine receptor expressed in many malignant cancer cells, induced MHC-I heavy chain down-regulation from the cell surface of the human epithelioid carcinoma HeLa cells, the human U251 and U87 glioblastoma cells, the human MDA-MD 231 breast cancer cells, and the human SK-N-BE (2) neuroblastoma cells. Activation of CXCR4 also induced MHC-I down-regulation in human peripheral blood mononuclear cells. The internalized MHC-I heavy chain molecules were partially co-localized with Rab7, a later endosomal marker. Activation of CXCR4 induced ubiquitination of MHC-I heavy chain, and mutation of the C-terminal two lysine residues (Lys-332, Lys-337) on one of the MHC-I alleles, HLA.B7, blocked CXCR4-evoked ubiquitination and down-regulation of HLA.B7. Moreover, purified GST-conjugated CXCR4 C terminus directly associated with the purified His-tagged beta2-microglobulin (beta2M), and MHC-I heavy chain was co-immunoprecipitated with CXCR4 in a beta2M-dependent manner. This interaction appears to be critical for CXCR4-evoked down-regulation of MHC-I heavy chain as evidenced by the data that MHC-I heavy chain down-regulation was inhibited by either truncation of the CXCR4 C terminus or knockdown of beta2M. All together, these findings shed new light on the role of CXCR4 in tumor evasion of immune surveillance via inducing MHC-I down-regulation from the cell surface.

[Immunological aspects of anticancer chemotherapy]. / Contribution du système immunitaire a l'efficacité des chimiothérapies anticancéreuses.

For over 40 years, four therapeutic modalities, namely surgery, radiotherapy, chemotherapy and hormone therapy have formed the core of anticancer treatments. Their mode of action is thought to involve a direct cytotoxic action on tumor cells. Recently, the discovery of tumor-associated immunosuppression and tumor immunosurveillance has led to cancer being reconsidered not only as an organ disease but also as a host disease. This new concept is supported by the recent discovery of the immunogenic effects of tumor cell death induced by a variety of cytotoxic drugs. This work describes a new pathway of tumor-derived antigen presentation mediated by the alarmin HMGB1 (released by dying tumor cells in response to chemo/radiotherapy) and by TLR4 on dendritic cells. In this model, TLR4 recognizes? tumor-derived antigens, leading to T cell activation and to the induction of an antitumor immune response. Accordingly, we show that breast cancer patients bearing a loss-of-function mutation of the TLR4 receptor have shorter disease-free survival, confirming the major role of the immune system in the response to cytotoxic treatments. The response to chemotherapy and/or radiotherapy may thus combine both direct cytotoxic effects and the development of long-term antitumor immunity. We anticipate that these new results will have major impact on cancer management.

Expression of high levels of human proteinase inhibitor 9 blocks both perforin/granzyme and Fas/Fas ligand-mediated cytotoxicity.

Proteinase inhibitor 9 (PI-9, SerpinB9) is the only known human intracellular granzyme B inhibitor. Whether expression of PI-9 is sufficient to block cytolysis induced by cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells remains controversial. To evaluate the roles of PI-9, we isolated and tested three lines of stably transfected HeLa cells expressing wild-type PI-9 and one line expressing an inactive mutant PI-9. Expressions of wild-type PI-9, but not the inactive mutant PI-9, inhibited cytolysis induced by human NK92 and NKL natural killer cells. Expression of high levels of PI-9 is therefore sufficient to protect human cells against NK cell-mediated cell death. Using two assays, we show that expressing wild-type PI-9, but not the inactive mutant PI-9, blocks Fas/Fas ligand (Fas/FasL)-mediated apoptosis. PI-9 expression has no effect on etoposide-induced apoptosis. HeLa cells exhibiting substantial resistance to Fas/FasL-mediated apoptosis contain 2- to 3-fold higher PI-9 levels than HCT116 human colon cancer cells and 2- to 3-fold lower PI-9 levels than MCF7/ERHA breast cancer cells, in which PI-9 is strongly induced by estrogens, and by tamoxifen. Expression of increasing levels of PI-9 in target cells may progressively inhibit immune surveillance by blocking NK and CTL-induced cytotoxicity through the perforin/granzyme pathway and then through the Fas/FasL pathway.

BeWo trophoblasts are unable to control replication of Toxoplasma gondii, even in the presence of exogenous IFN-gamma.

The ability of RH strain of Toxoplasma gondii to invade and grow into BeWo cells was investigated in the present study using IFN-gamma, l-tryptophan, or alpha-methyl-tryptophan treatments. HeLa cells were used in the same conditions for comparison purposes. It was demonstrated that BeWo cells are more permissive to T. gondii infection, making them more susceptible to this pathogen when compared to HeLa cells. Infection rates of BeWo cells do not show any significant alteration in different protocols using IFN-gamma. In addition, BeWo treated with l-tryptophan was unable to significantly increase parasite growth. In contrast, HeLa cells treated with IFN-gamma or IFN-gamma plus l-tryptophan are able to impair or increase, respectively, parasite replication, providing evidence that this indoleamine-2,3-dioxygenase-dependent phenomenon is operant in these cells, whereas it is inactive in BeWo. Therefore, our data support the hypothesis that the immunological mechanisms controlling infection at the maternal-fetal interface are different from those occurring in the periphery. At the same time that operating regulatory mechanisms work inside and outside the cells located at that microenvironment to prevent maternal rejection of the concept, these events might facilitate the progression of infection caused by intracellular pathogens, as T. gondii.

Salmonella pathogenicity island 2 is expressed prior to penetrating the intestine.

Salmonella enterica serovar Typhimurium is a facultative intracellular pathogen that causes disease in mice that resembles human typhoid. Typhoid pathogenesis consists of distinct phases in the intestine and a subsequent systemic phase in which bacteria replicate in macrophages of the liver and spleen. The type III secretion system encoded by Salmonella pathogenicity island 2 (SPI-2) is a major virulence factor contributing to the systemic phase of typhoid pathogenesis. Understanding how pathogens regulate virulence mechanisms in response to the environment, including different host tissues, is key to our understanding of pathogenesis. A recombinase-based in vivo expression technology system was developed to assess SPI-2 expression during murine typhoid. SPI-2 expression was detectable at very early times in bacteria that were resident in the lumen of the ileum and was independent of active bacterial invasion of the epithelium. We also provide direct evidence for the regulation of SPI-2 by the Salmonella transcription factors ompR and ssrB in vivo. Together these results demonstrate that SPI-2 expression precedes penetration of the intestinal epithelium. This induction of expression precedes any documented SPI-2-dependent phases of typhoid and may be involved in preparing Salmonella to successfully resist the antimicrobial environment encountered within macrophages.

Chlamydia trachomatis variant with nonfusing inclusions: growth dynamic and host-cell transcriptional response.

We compared growth rate and host-cell transcriptional responses of a Chlamydia trachomatis variant strain and a prototype strain. Growth dynamics were estimated by 16S rRNA level and by inclusion-forming units (IFUs) at different times after infection in HeLa cells. When inoculated at the same multiplicity of infection and observed 24-48 h after infection, the variant 16S rRNA transcriptional level was 3%-4% that of the prototype, and the IFUs of the variant strain were 0.1%-1% those of the prototype. Specific host-cell transcriptional responses to the variant were identified in a global-expression microarray in which variant strain-infected cells were compared with mock-infected and prototype strain-infected cells. In variant strain-infected cells, 47% (16/34) of specifically induced host genes were related to immunity and 32% (8/25) of specifically suppressed genes were related to lipid metabolism. The variant strain grew significantly more slowly and induced a modified host-cell transcriptional response, compared with the prototype strain.

Generation of novel covalent RNA-protein complexes in cells by ultraviolet B irradiation: implications for autoimmunity.

OBJECTIVE: To determine whether ultraviolet B (UVB) irradiation induces novel modifications in autoantigens targeted during experimental photoinduced epidermal damage. METHODS: To search for novel UVB-induced autoantigen modifications, lysates made from UVB-irradiated human keratinocytes or HeLa cells were immunoblotted using human autoantibodies that recognize ribonucleoprotein autoantigens. Novel autoantigen structures identified were further characterized using nucleases and RNA hybridization. RESULTS: Human sera that recognize U1-70 kd (U1-70K) and La by immunoblotting also recognized multiple novel species when they were used to immunoblot lysates of UVB-irradiated keratinocytes or HeLa cells. These species were not present in control cells and were not observed when apoptosis was induced by Fas ligation or cytotoxic lymphocyte granule contents. Biochemical analysis using multiple assays revealed that these novel UVB-induced molecular species result from the covalent crosslinking between the U1 RNA and the hYRNA molecules with their associated proteins, including U1-70K, La, and likely components of the Sm particle. CONCLUSION: These data demonstrate that UVB irradiation of live cells can directly induce covalent RNA-protein complexes, which are recognized by human autoantibodies. As previously described for other autoantigens, these covalent complexes of RNA and proteins may have important consequences in terms of antigen capture and processing.

Safety characterization of HeLa-based cell substrates used in the manufacture of a recombinant adeno-associated virus-HIV vaccine.

The use of transformed cell substrates for prophylactic vaccine manufacturing is widely debated. Extensive characterization is required to address the suitability of neoplastic cell substrates for vaccine manufacture. The HeLa-based cell substrate used in the manufacture of a prophylactic rAAV-HIV vaccine, AAV2-gagPR delta RT (tgAAC09) was tested in vivo for its tumor-forming potential, the oncogenic potential of its high molecular weight DNA and the potential presence of occult oncogenic adventitious agents. This data from these in vivo studies, in conjunction with prion gene and protein characterization, cell and viral clearance studies and quantity of residual host-cell DNA levels in the purified tgAAC09 vaccine, were used to establish what we believe to be an acceptable safety profile for the vaccine manufacturing process. The tumor-producing dose in 50% of the animals was consistent with that in a published report from FDA staff for HeLa cells. High molecular weight cellular DNA was not oncogenic and no occult oncogenic agents were detected by testing in nude mice and newborn rodent models, respectively. Endogenous prion protein was also normal and genomic sequence analysis detected no mutations associated with increased risk of prion disease. In addition, the purification process used to produce this vaccine candidate removed all detectable cells (clearance of greater than 22 log10), viral clearance study showed 6-17 log10 clearance of three model viruses and host-cell DNA in the bulk product was less than 100pg host-cell DNA per dose of 3 x 10(11) DNase resistant particles (DRP) of the vaccine. Taken together, the data from the in vivo and in vitro tests that were performed to characterize the HeLa based producer cell line (T3B12-5B) and HeLa S3 cells support the use of these cells as substrates for the manufacture of a purified rAAV-HIV vaccine candidate. The data also supports the ability of the process, employing the HeLa cell substrate, used to manufacture the rAAV-HIV vaccine to produce a product as free of adventitious agents as current testing procedures can document. Safety of the rAAV-HIV vaccine is currently being assessed in a Phase I clinical trial.

Presence of autoantibodies against HeLa small nuclear ribonucleoproteins in chagasic and non-chagasic cardiac patients.

We detected anti-human small nuclear ribonucleoprotein (snRNP) autoantibodies in chagasic patients by different immunological methods using HeLa snRNPs. ELISA with Trypanosoma cruzi total lysate antigen or HeLa human U small nuclear ribonucleoproteins (UsnRNPs) followed by incubation with sera from chronic chagasic and non-chagasic cardiac patients was used to screen and compare serum reactivity. Western blot analysis using a T. cruzi total cell extract was also performed in order to select some sera for Western blot and immunoprecipitation assays with HeLa nuclear extract. ELISA showed that 73 and 95% of chronic chagasic sera reacted with HeLa UsnRNPs and T. cruzi antigens, respectively. The Western blot assay demonstrated that non-chagasic cardiac sera reacted with high molecular weight proteins present in T. cruzi total extract, probably explaining the 31% reactivity found by ELISA. However, these sera reacted weakly with HeLa UsnRNPs, in contrast to the chagasic sera, which showed autoantibodies with human Sm (from Stefanie Smith, the first patient in whom this activity was identified) proteins (B/B', D1, D2, D3, E, F, and G UsnRNP). Immunoprecipitation reactions using HeLa nuclear extracts confirmed the reactivity of chagasic sera and human UsnRNA/RNPs, while the other sera reacted weakly only with U1snRNP. These findings agree with previously reported data, thus supporting the idea of the presence of autoimmune antibodies in chagasic patients. Interestingly, non-chagasic cardiac sera also showed reactivity with T. cruzi antigen and HeLa UsnRNPs, which suggests that individuals with heart disease of unknown etiology may develop autoimmune antibodies at any time. The detection of UsnRNP autoantibodies in chagasic patients might contribute to our understanding of how they develop upon initial T. cruzi infection.

Perfil de auto-anticorpos na surdez súbita, surdez rapidamente progressiva e doença de Ménière / Autoantibody profile in instantaneous hearing loss, rapidly progressive hearing loss and Ménière disease

A surdez neurossensorial (SNS) imunomediada é uma das formas de SNS com possibilidade de reversão. Surdez súbita, surdez rapidamente progressiva e doença de Ménière são relacionadas à etiologia autoimune. O Western blot (WB) com antígeno de tecidos bovinos é usado para detecção do anticorpo anti-68kD (hsp70) em SNS imunomediada. Marcadores específicos são necessários para diagnóstico e prognóstico desta entidade. O objetivo do estudo foi determinar reatividade dos soros de pacientes com SNS contra antígeno de células humanas (HeLa) pelo WB. Observou-se reatividade contra células HeLa, confirmando a presença de autorreatividade e autoanticorpos. O papel destes anticorpos ainda é desconhecido. /Immune-mediated sensorineural hearing loss (SNHL) is one of few forms of reversible SNHL. The rapidly progressive hearing loss, sudden SNHL and Ménière’s disease are often related to autoimmune etiology. Western blot with bovine tissues as target has been used, detecting anti-68kD antibody (hsp70) in immune-mediated SNHL. Other specific markers are necessary to diagnosis and prognosis. The aim of this study was determinate reactivity from SNHL patients’ sera against human cell line antigen (HeLa) by Western blot. Reactivity to HeLa cells was observed reinforcing autoreactivity and autoantibody presence. The role of these autoantibodies is unknown and futher studies are necessary...

Cytolethal distending toxin: a potential virulence factor for Helicobacter cinaedi.

Although Helicobacter cinaedi is the most commonly reported enterohepatic helicobacter in humans, there are no reports of virulence factors, and little is known about how it infects and causes disease. In this study, H. cinaedi isolates from humans and animals were examined for production of cytolethal distending toxin (Cdt). Cdt causes distention in cells and arrest in the G2/M phase of cell division. It is encoded by three genes: cdtA, cdtB, and cdtC. cdtB is the most conserved. All isolates except the American Type Culture Collection strain (identified as H. fennelliae) demonstrated Cdt activity and had a cdtB polymerase chain-reaction product homologous with known cdtB. Deduced amino acid sequences of cdtB showed a high (93%-99%) degree of similarity among the H. cinaedi isolates. H. cinaedi shares the production of Cdt with other enteric pathogens, including enterohepatic Helicobacter species. This is the first report of a putative virulence factor in H. cinaedi.

Genetic modifications of the adeno-associated virus type 2 capsid reduce the affinity and the neutralizing effects of human serum antibodies.

The high prevalence of human serum antibodies against adeno-associated virus type 2 (AAV) vectors represents a potential limitation for in vivo applications. Consequently, the development of AAV vectors able to escape antibody binding and neutralization is of importance. To identify capsid domains which contain major immunogenic epitopes, six AAV capsid mutants carrying peptide insertions in surface exposed loop regions (I-261, I-381, I-447, I-534, I-573, I-587) were analyzed. Two of these mutants, I-534 and I-573, showed an up to 70% reduced affinity for AAV antibodies as compared to wild-type AAV in the majority of serum samples. In addition, AAV mutant I-587 but not wild-type AAV efficiently transduced cells despite the presence of neutralizing antisera. Taken together, the results show that major neutralizing effects of human AAV antisera might be overcome by the use of AAV capsid mutants.