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.

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.

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.

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...

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.

Identification of a naturally processed HLA-A*0201 HPV18 E7 T cell epitope by tumor cell mediated in vitro vaccination.

Immunotherapy of HPV-associated disease such as cervical cancer is moving from preclinical investigation to clinical trials. The viral oncoproteins E6 and E7 are ideal target antigens because their expression is mandatory in HPV-transformed tumor cells. T cells are the most important effector cells for therapeutic vaccination strategies. Therefore, the identification and characterization of HPV E6 and E7 T cell epitopes is necessary. Methods to date rely on screening for immunogenicity of peptides predicted by algorithms. Presentation of the identified peptides on tumor cells, however, needs to be confirmed. In our study, we have improved the method to identify peptide epitopes of HPV18 E7 that are actually presented by tumor cells. We induced allogeneic T-cell lines by stimulation with HPV18-positive, CD80 and HLA-A*0201 transfected cervical cancer cells. Sensitized T cells were probed against an array of a HPV18 E7 20mer peptide-library. We found specific reactivity to one of the 20mer peptides. This sequence was then screened via algorithms for putative epitopes. One putative HLA-A2 restricted epitope was confirmed to bind to HLA-A2, to be immunogenic and to induce IFN gamma-release in ELISpot assays. Epitope-specific T cells were cytolytic toward autologous peptide pulsed targets and HPV18 transformed tumor cells. The identification of epitope-specific T cells in tumor infiltrating lymphocytes of a HPV18-positive HLA-matched cervical cancer patient suggests an in vivo relevance of the identified epitope. We suggest that our approach is advantageous over conventional methods, because it yields candidate peptides that are relevant CTL epitopes that are expressed, processed and presented by tumor cells.

Rational design of a CD4 mimic that inhibits HIV-1 entry and exposes cryptic neutralization epitopes.

The conserved surfaces of the human immunodeficiency virus (HIV)-1 envelope involved in receptor binding represent potential targets for the development of entry inhibitors and neutralizing antibodies. Using structural information on a CD4-gp120-17b antibody complex, we have designed a 27-amino acid CD4 mimic, CD4M33, that presents optimal interactions with gp120 and binds to viral particles and diverse HIV-1 envelopes with CD4-like affinity. This mini-CD4 inhibits infection of both immortalized and primary cells by HIV-1, including primary patient isolates that are generally resistant to inhibition by soluble CD4. Furthermore, CD4M33 possesses functional properties of CD4, including the ability to unmask conserved neutralization epitopes of gp120 that are cryptic on the unbound glycoprotein. CD4M33 is a prototype of inhibitors of HIV-1 entry and, in complex with envelope proteins, a potential component of vaccine formulations, or a molecular target in phage display technology to develop broad-spectrum neutralizing antibodies.

HeLa cells cocultured with peripheral blood lymphocytes acquire an immuno-inhibitory phenotype through up-regulation of indoleamine 2,3-dioxygenase activity.

The mechanisms by which tumour cells escape recognition by the immune system or subvert antitumour effector responses remain poorly understood. In the course of investigating the potential of costimulatory signals in anticancer immunotherapy strategies, we have observed that HeLa cells (a human cervical carcinoma cell line) cocultured with peripheral blood lymphocytes (PBL) acquire the capacity to inhibit PBL proliferation in response to interleukin-2 (IL-2). This immuno-inhibitory phenotype was further shown to result from induction of the tryptophan-catabolizing enzyme, indoleamine 2,3-dioxygenase (IDO), by interferon-gamma (IFN-gamma) secreted from cocultured allo-reactive PBL. This enzyme has recently been shown to be a critically important modulator of immunological responses, most notably through the capacity to protect allogeneic concepti from alloreactive maternal lymphocytes. While the cytostatic consequences of IDO activity in tumour cells has received attention, the data presented in this report support the hypothesis that IDO activity may also act to impair antitumour immune responses.

Inhibition of mitogen-activated protein kinase signaling by chloroquine.

Previously, we demonstrated that the anti-inflammatory drug chloroquine (CQ) inhibited LPS-induced TNF-alpha transcription. To define further the mechanism of CQ, we studied the effect of this drug on mitogen-activated protein kinase signaling pathways involved in regulation of TNF production. CQ interfered with phosphorylation of extracellular signal-regulated kinases (ERK)1/2 and the ERK-activating kinases mitogen-activating protein/ERK kinase (MEK)1/2. Both CQ and PD98059, a MEK1 inhibitor, reduced luciferase reporter activity driven by human TNF promoter sequences. However, CQ appeared to mediate these effects by deactivating Raf, the upstream activator of MEK. These findings were supported by functional data demonstrating that CQ and PD98059 interfered with TNF expression in several human and murine cell types while neither inhibitor blocked TNF production in murine RAW264.7 macrophages, a cell line that does not require MEK-ERK signaling for TNF production. Finally, we evaluated whether CQ could sensitize HeLa cells to undergo anti-Fas-mediated apoptosis, an effect observed when ERK activation is interrupted in this cell line. CQ rendered HeLa cells sensitive to anti-Fas treatment in a manner similar to PD98059. Taken together, these data argue that therapeutic concentrations of CQ interfere with ERK activation by a novel mechanism, an effect that could be responsible, at least in part, for the potent anti-inflammatory effects of this drug.

A proinflammatory role for the cyclopentenone prostaglandins at low micromolar concentrations: oxidative stress-induced extracellular signal-regulated kinase activation without NF-kappa B inhibition.

An anti-inflammatory role and therapeutic potential for cyclopentenone PGs (cyPGs) has been suggested, based on observations that levels of cyPGs in exudates increase during the resolution phase of inflammation, and that exogenous cyPGs may attenuate the inflammatory response in vivo and in vitro mainly through inhibition of NF-kappaB, a critical activator of inflammatory gene expression. However, exogenous cyPGs inhibit NF-kappaB only at concentrations substantially higher than those of endogenous cyPGs present in inflammatory fluids, thus challenging the hypothesis that cyPGs are naturally occurring inhibitors of inflammation and suggesting that cyPGs at low concentrations might have previously unappreciated effects. In this study, using various cell types, we report that cyPGs, when used at concentrations substantially lower than required for NF-kappaB inhibition (viz, low micromolar concentrations), significantly potentiate the inflammatory response to TNF-alpha. At these concentrations, cyPGs induce production of reactive oxygen species, thereby synergizing with TNF-alpha to activate the extracellular signal-regulated kinase 1/2, an activation which in turn potentiates proinflammatory cytokine expression at both transcriptional and posttranscriptional levels. Our study establishes a proinflammatory role for cyPGs at low micromolar concentrations, raises the possibility that cyPGs do not act as physiologic anti-inflammatory mediators, and questions the therapeutic potential of these compounds.

Enhanced tumor cell selectivity of adriamycin-monoclonal antibody conjugate via a poly(ethylene glycol)-based cleavable linker.

A novel linker consisting of poly(ethylene glycol) (PEG) and dipeptide was used for conjugation of adriamycin with tumor-specific monoclonal antibody, NL-1, to confirm that the linker can be cleaved selectively with the tumor specific enzyme to express cytotoxicity of the anti-tumor agent. Initially, adriamycin-conjugated PEG linkers through different amino acid compositions, alanyl-valine (Ala-Val), alanyl-proline (Ala-Pro), and glycyl-proline (Gly-Pro) sequences, were prepared to confirm selective digestion with model enzymes. Adriamycin was released by particular model endoproteases, thermolysin and proline endopeptidase, from the linkers with different efficiency. When conjugates were prepared using these adriamycin-bound linkers, conjugates had no loss of binding affinity and specificity for common acute lymphoblastic leukemia antigen (CALLA) expressed on the Daudi cell surfaces as the target of NL-1 antibody. In addition, adriamycin release from the conjugates was also confirmed by incubating them with specific proteases. Tumor cell growth was inhibited dose-dependently for the conjugates carrying Ala-Val and Gly-Pro linkers, whereas significant inhibitory effect was abolished for the conjugate carrying Ala-Pro linker, indicating that cytotoxic effect can be controlled by specificity of antibody and composition of linker peptide. IC(50) for Ala-Val linked conjugate was approximately 3.5 microg/ml and that for Gly-Pro linked conjugate was 5.2 microg/ml. PEG-dipeptidyl linker demonstrated here will be an effective tool for the preparation of immunoconjugate, especially specific activation of anti-tumor agents at desired tumor tissues.

[Specific anti-tumor effect of immune competent cell induced by HSP-70 of HeLa cells in vitro].

BACKGROUND & OBJECTIVE: Recent studies indicated that heat shock proteins (HSPs) are associated with antigenic peptides and participate in the antitumor T cell response in animals. This study was designed to investigate the mechanism of anti-tumor effect of immune competent cells (ICC) induced by HSP-70 of HeLa (cervical carcinoma) cells in vitro. METHODS: The normal peripheral blood mononuclear cells(PBMCs) stimulated with HSP-70 of HeLa cells in vitro. T cell phenotypes were analyzed before and after stimulated by PBMCs. HSP-70 primed the immune competent cells (ICCs) from PBMCs were tested for cytotoxic activity against HeLa cell. RESULTS: CD3+ cell was 85.73 +/- 1.44%, up from 57.68 +/- 1.46% before the amplification(P < 0.002). CD8+ cell was 48.06 +/- 1.66%, up from 23.56 +/- 1.86% before the amplification(P < 0.002). The ICCs induced by HSP-70 were shown significant cytotoxic activity (82.69 +/- 1.97%, 62.11 +/- 1.61%) against HeLa cells and cervical carcinoma cells, and the cytotoxic activity (31.05 +/- 2.09%) of the ICC against HeLa cells could be blocked by anti-HSP-70 antibody. CONCLUSION: The ICC can be induced selectively by PBMCs with HSP-70 of HeLa cells, and has specific cytotoxic activity against HeLa cell.

Novel mechanism of antibody-independent complement neutralization of herpes simplex virus type 1.

The envelope surface glycoprotein C (gC) of HSV-1 interferes with the complement cascade by binding C3 and activation products C3b, iC3b, and C3c, and by blocking the interaction of C5 and properdin with C3b. Wild-type HSV-1 is resistant to Ab-independent complement neutralization; however, HSV-1 mutant virus lacking gC is highly susceptible to complement resulting in > or =100-fold reduction in virus titer. We evaluated the mechanisms by which complement inhibits HSV-1 gC null virus to better understand how gC protects against complement-mediated neutralization. C8-depleted serum prepared from an HSV-1 and -2 Ab-negative donor neutralized gC null virus comparable to complement-intact serum, indicating that C8 and terminal lytic activity are not required. In contrast, C5-depleted serum from the same donor failed to neutralize gC null virus, supporting a requirement for C5. EDTA-treated serum did not neutralize gC null virus, indicating that complement activation is required. Factor D-depleted and C6-depleted sera neutralized virus, suggesting that the alternative complement pathway and complement components beyond C5 are not required. Complement did not aggregate virus or block attachment to cells. However, complement inhibited infection before early viral gene expression, indicating that complement affects one or more of the following steps in virus replication: virus entry, uncoating, DNA transport to the nucleus, or immediate early gene expression. Therefore, in the absence of gC, HSV-1 is readily inhibited by complement by a C5-dependent mechanism that does not require viral lysis, aggregation, or blocking virus attachment.