Delivery of self-amplifying RNA vaccines in in vitro reconstituted virus-like particles.

Many mRNA-based vaccines have been investigated for their specific potential to activate dendritic cells (DCs), the highly-specialized antigen-presenting cells of the immune system that play a key role in inducing effective CD4+ and CD8+ T-cell responses. In this paper we report a new vaccine/gene delivery platform that demonstrates the benefits of using a self-amplifying ("replicon") mRNA that is protected in a viral-protein capsid. Purified capsid protein from the plant virus Cowpea Chlorotic Mottle Virus (CCMV) is used to in vitro assemble monodisperse virus-like particles (VLPs) containing reporter proteins (e.g., Luciferase or eYFP) or the tandem-repeat model antigen SIINFEKL in RNA gene form, coupled to the RNA-dependent RNA polymerase from the Nodamura insect virus. Incubation of immature DCs with these VLPs results in increased activation of maturation markers - CD80, CD86 and MHC-II - and enhanced RNA replication levels, relative to incubation with unpackaged replicon mRNA. Higher RNA uptake/replication and enhanced DC activation were detected in a dose-dependent manner when the CCMV-VLPs were pre-incubated with anti-CCMV antibodies. In all experiments the expression of maturation markers correlates with the RNA levels of the DCs. Overall, these studies demonstrate that: VLP protection enhances mRNA uptake by DCs; coupling replicons to the gene of interest increases RNA and protein levels in the cell; and the presence of anti-VLP antibodies enhances mRNA levels and activation of DCs in vitro. Finally, preliminary in vivo experiments involving mouse vaccinations with SIINFEKL-replicon VLPs indicate a small but significant increase in antigen-specific T cells that are doubly positive for IFN and TFN induction.

Maximizing in vivo target clearance by design of pH-dependent target binding antibodies with altered affinity to FcRn.

Antibodies with pH-dependent binding to both target antigens and neonatal Fc receptor (FcRn) provide an alternative tool to conventional neutralizing antibodies, particularly for therapies where reduction in antigen level is challenging due to high target burden. However, the requirements for optimal binding kinetic framework and extent of pH dependence for these antibodies to maximize target clearance from circulation are not well understood. We have identified a series of naturally-occurring high affinity antibodies with pH-dependent target binding properties. By in vivo studies in cynomolgus monkeys, we show that pH-dependent binding to the target alone is not sufficient for effective target removal from circulation, but requires Fc mutations that increase antibody binding to FcRn. Affinity-enhanced pH-dependent FcRn binding that is double-digit nM at pH 7.4 and single-digit nM at pH 6 achieved maximal target reduction when combined with similar target binding affinities in reverse pH directions. Sustained target clearance below the baseline level was achieved 3 weeks after single-dose administration at 1.5 mg/kg. Using the experimentally derived mechanistic model, we demonstrate the essential kinetic interplay between target turnover and antibody pH-dependent binding during the FcRn recycling, and identify the key components for achieving maximal target clearance. These results bridge the demand for improved patient dosing convenience with the "know-how" of therapeutic modality by design.

Efficient Qualitative and Quantitative Determination of Antigen-induced Immune Responses.

To determine the effectiveness of immunization strategies used in therapeutic antibody or vaccine development, it is critical to assess the quality of immunization-induced polyclonal antibody responses. Here, we developed a workflow that uses sensitive methods to quantitatively and qualitatively assess immune responses against foreign antigens with regard to antibody binding affinity and epitope diversity. The application of such detailed assessments throughout an immunization campaign can significantly reduce the resources required to generate highly specific antibodies. Our workflow consists of the following two steps: 1) the use of surface plasmon resonance to quantify antigen-specific antibodies and evaluate their apparent binding affinities, and 2) the recovery of serum IgGs using an automated small scale purification system, followed by the determination of their epitope diversity using hydrogen deuterium exchange coupled with mass spectrometry. We showed that these methods were sensitive enough to detect antigen-specific IgGs in the nanogram/µl range and that they provided information for differentiating the antibody responses of the various immunized animals that could not be obtained by conventional methods. We also showed that this workflow can guide the selection of an animal that produces high affinity antibodies with a desired epitope coverage profile, resulting in the generation of potential therapeutic monoclonal antibody clones with desirable functional profiles. We postulate that this workflow will be an important tool in the development of effective vaccines to combat the highly sophisticated evasion mechanisms of pathogens.

Immunogenicity of a prime-boost vaccine containing the circumsporozoite proteins of Plasmodium vivax in rodents.

Plasmodium vivax is the most widespread and the second most prevalent malaria-causing species in the world. Current measures used to control the transmission of this disease would benefit from the development of an efficacious vaccine. In the case of the deadly parasite P. falciparum, the recombinant RTS,S vaccine containing the circumsporozoite antigen (CSP) consistently protects 30 to 50% of human volunteers against infection and is undergoing phase III clinical trials in Africa with similar efficacy. These findings encouraged us to develop a P. vivax vaccine containing the three circulating allelic forms of P. vivax CSP. Toward this goal, we generated three recombinant bacterial proteins representing the CSP alleles, as well as a hybrid polypeptide called PvCSP-All-CSP-epitopes. This hybrid contains the conserved N and C termini of P. vivax CSP and the three variant repeat domains in tandem. We also generated simian and human recombinant replication-defective adenovirus vectors expressing PvCSP-All-CSP-epitopes. Mice immunized with the mixture of recombinant proteins in a formulation containing the adjuvant poly(I·C) developed high and long-lasting serum IgG titers comparable to those elicited by proteins emulsified in complete Freund's adjuvant. Antibody titers were similar in mice immunized with homologous (protein-protein) and heterologous (adenovirus-protein) vaccine regimens. The antibodies recognized the three allelic forms of CSP, reacted to the repeated and nonrepeated regions of CSP, and recognized sporozoites expressing the alleles VK210 and VK247. The vaccine formulations described in this work should be useful for the further development of an anti-P. vivax vaccine.

Correlates of relative resistance against low-dose rectal simian immunodeficiency virus challenges in peripheral blood mononuclear cells of vaccinated rhesus macaques.

In this study, we compared the immunogenicity and protection from repeated low-dose intrarectal SIVmac251 challenge in two groups of vaccinated RMs. Animals were immunized with live SIVmac239, which had been attenuated by a deletion of the nef sequence, or they were vaccinated twice with an E1-deleted AdHu5, expressing SIVmac239gag. The vaccinated animals and a cohort of unvaccinated control animals were then challenged 10 times in weekly intervals with low doses of SIVmac251 given rectally. Our results confirm previous studies showing that whereas SIVΔnef provides some degree of protection against viral acquisition after repeated low-dose rectal SIVmac251 challenges, vaccination with an AdHu5gag vaccine designed to induce only antiviral T cell responses is ineffective. As immunological analyses of prechallenge, vaccine-induced T and B cell responses failed to reveal correlates of protection that distinguished the more susceptible from the more resistant vaccinated animals, we carried out RNA-Seq studies of paired pre- and postvaccination samples to identify transcriptional patterns that correlated with the differences in response. We show that gene expression signatures associated with the delayed SIV infection seen in some AdHu5gag recipients were largely present in prevaccination samples of those animals. In contrast, the responding SIVΔnef-immunized animals showed a predominance of vaccine-induced changes, thus enabling us to define inherited and vaccine-induced gene expression signatures and their associated pathways that may play a role in preventing SIV acquisition.

Capsid-specific T-cell responses to natural infections with adeno-associated viruses in humans differ from those of nonhuman primates.

Hepatic adeno-associated virus serotype 2 (AAV2)-mediated gene transfer failed to achieve sustained transgene product expression in human subjects. We formulated the hypothesis that rejection of AAV-transduced hepatocytes is caused by AAV capsid-specific CD8(+) T cells that become reactivated upon gene transfer. Although this hypothesis was compatible with clinical data, which showed a rise in circulating AAV capsid-specific T cells following injection of AAV vectors, it did not explain that AAV vectors achieved long-term transgene expression in rhesus macaques, which are naturally infected with AAV serotypes closely related to those of humans. To address this apparent contradiction, we tested human and rhesus macaque samples for AAV capsid-specific T cells by intracellular cytokine staining combined with staining for T-cell subset and differentiation markers. This highly sensitive method, which could provide a tool to monitor adverse T-cell responses in gene transfer trials, showed that AAV capsid-specific CD8(+) and CD4(+) T cells can be detected in blood of naturally infected humans and rhesus macaques. They are present at higher frequencies in rhesus macaques. Furthermore, T cells from humans and rhesus macaques exhibit striking differences in their differentiation status and in their functions, which may explain the disparate duration of AAV-mediated gene transfer in these two species.

Active immunotherapy combined with blockade of a coinhibitory pathway achieves regression of large tumor masses in cancer-prone mice.

Vaccines that aim to expand tumor-specific CD8(+) T cells have yielded disappointing results in cancer patients although they showed efficacy in transplantable tumor mouse models. Using a system that more faithfully mimics a progressing cancer and its immunoinhibitory microenvironment, we here show that in transgenic mice, which gradually develop adenocarcinomas due to expression of HPV-16 E7 within their thyroid, a highly immunogenic vaccine expressing E7 only induces low E7-specific CD8(+) T-cell responses, which fail to affect the size of the tumors. In contrast, the same type of vaccine expressing E7 fused to herpes simplex virus (HSV)-1 glycoprotein D (gD), an antagonist of the coinhibitory B- and T-lymphocyte attenuator (BTLA)/CD160-herpes virus entry mediator (HVEM) pathways, stimulates potent E7-specific CD8(+) T-cell responses, which can be augmented by repeated vaccination, resulting in initial regression of even large tumor masses in all mice with sustained regression in more than half of them. These results indicate that active immunization concomitantly with blockade of the immunoinhibitory HVEM-BTLA/CD160 pathways through HSV-1 gD may result in sustained tumor regression.

Vaccine-induced T cells provide partial protection against high-dose rectal SIVmac239 challenge of rhesus macaques.

Despite enormous efforts by the scientific community, an effective HIV vaccine remains elusive. To further address to what degree T cells in absence of antibodies may protect against simian immunodeficiency virus (SIV) disease progression, rhesus macaques were vaccinated intramuscularly with a chimpanzee-derived Ad vector (AdC) serotype 6 and then boosted intramuscularly with a serologically distinct AdC vector of serotype 7 both expressing Gag of SIVmac239. Animals were subsequently boosted intramuscularly with a modified vaccinia Ankara (MVA) virus expressing Gag and Tat of the homologous SIV before mucosal challenge with a high dose of SIVmac239 given rectally. Whereas vaccinated animals showed only a modest reduction of viral loads, their overall survival was improved, in association with a substantial protection from the loss of CD4(+) T cells. In addition, the two vaccinated Mamu-A*01(+) macaques controlled viral loads to levels below detection within weeks after challenge. These data strongly suggest that T cells, while unable to affect SIV acquisition upon high-dose rectal infection, can reduce disease progression. Induction of potent T-cell responses should thus remain a component of our efforts to develop an efficacious vaccine to HIV-1.

A universal influenza A vaccine based on adenovirus expressing matrix-2 ectodomain and nucleoprotein protects mice from lethal challenge.

A universal influenza vaccine, designed to induce broadly cross-reactive immunity against current and future influenza A virus strains, is in critical demand to reduce the need for annual vaccinations with vaccines chosen upon predicting the predominant circulating viral strains, and to ameliorate the threat of cyclically occurring pandemics that have, in the past, killed tens of millions. Here, we describe a vaccine regimen based on sequential immunization with two serologically distinct chimpanzee-derived replication-defective adenovirus (Ad) vectors expressing the matrix-2 protein ectodomain (M2e) from three divergent strains of influenza A virus fused to the influenza virus nucleoprotein (NP) for induction of antibodies to M2e and virus-specific CD8(+) T cells to NP. In preclinical mouse models, the Ad vaccines expressing M2e and NP elicit robust NP-specific CD8(+) T-cell responses and moderate antibody responses to all three M2e sequences. Most importantly, vaccinated mice are protected against morbidity and mortality following challenge with high doses of different influenza virus strains. Protection requires both antibodies to M2e and cellular immune responses to NP.

Immune responses and therapeutic antitumor effects of an experimental DNA vaccine encoding human papillomavirus type 16 oncoproteins genetically fused to herpesvirus glycoprotein D.

Recombinant adenovirus or DNA vaccines encoding herpes simplex virus type 1 (HSV-1) glycoprotein D (gD) genetically fused to human papillomavirus type 16 (HPV-16) oncoproteins (E5, E6, and E7) induce antigen-specific CD8(+) T-cell responses and confer preventive resistance to transplantable murine tumor cells (TC-1 cells). In the present report, we characterized some previously uncovered aspects concerning the induction of CD8(+) T-cell responses and the therapeutic anticancer effects achieved in C57BL/6 mice immunized with pgD-E7E6E5 previously challenged with TC-1 cells. Concerning the characterization of the immune responses elicited in mice vaccinated with pgD-E7E6E5, we determined the effect of the CD4(+) T-cell requirement, longevity, and dose-dependent activation on the E7-specific CD8(+) T-cell responses. In addition, we determined the priming/boosting properties of pgD-E7E6E5 when used in combination with a recombinant serotype 68 adenovirus (AdC68) vector encoding the same chimeric antigen. Mice challenged with TC-1 cells and then immunized with three doses of pgD-E7E6E5 elicited CD8(+) T-cell responses, measured by intracellular gamma interferon (IFN-γ) and CD107a accumulation, to the three HPV-16 oncoproteins and displayed in vivo antigen-specific cytolytic activity, as demonstrated with carboxyfluorescein diacetate succinimidyl ester (CFSE)-labeled target cells pulsed with oligopeptides corresponding to the H-2D(b)-restricted immunodominant epitopes of the E7, E6, or E5 oncoprotein. Up to 70% of the mice challenged with 5 × 10(5) TC-1 cells and immunized with pgD-E7E6E5 controlled tumor development even after 3 days of tumor cell challenge. In addition, coadministration of pgD-E7E6E5 with DNA vectors encoding pGM-CSF or interleukin-12 (IL-12) enhanced the therapeutic antitumor effects for all mice challenged with TC-1 cells. In conclusion, the present results expand our previous knowledge on the immune modulation properties of the pgD-E7E6E5 vector and demonstrate, for the first time, the strong antitumor effects of the DNA vaccine, raising promising perspectives regarding the development of immunotherapeutic reagents for the control of HPV-16-associated tumors.

Targeting inhibitory pathways in cancer immunotherapy.

The clinical success of adaptive transfer of in vitro expanded antigen-specific CD8(+) T cells isolated from patients' tumors has demonstrated that effector cells of the adaptive immune system can effectively eliminate even large tumor masses. Nevertheless, cancer vaccines that aim to expand such CD8(+) T cells in situ have had remarkably little success in spite of numerous attempts. Recent advances in basic immunology have revealed layers of complexity controlling activation and maintenance of adaptive immune responses that are tightly controlled by immunoinhibitory pathways to avoid horror autotoxicus. During tumor progression the activities of negative pathways increase and together with cancer immune evasion tactics presumably prevent induction of an efficacious immune response by cancer vaccines that solely provide more antigen to an already suppressed system. Cancer vaccines may thus need to readjust the imbalance of the cancer patients' immune system by inhibiting immunoinhibitors; such regimens have shown preclinical efficacy and are now entering clinical trials hopefully ending the Kafkaesque futility of cancer vaccines.

Augmentation of primary influenza A virus-specific CD8+ T cell responses in aged mice through blockade of an immunoinhibitory pathway.

Immune responses diminish with age resulting in an increased susceptibility of the elderly to infectious agents and an inability to mount protective immune responses to vaccines. Immunosenescence affects multiple aspects of the immune system, including CD8(+) T cells, which control viral infections and are assumed to prevent the development of cancers. In this study, we tested if CD8(+) T cell responses in aged mice could be enhanced through a vaccine that concomitantly expresses Ag and a molecule that blocks an immunoinhibitory pathway. Specifically, we tested a vaccine based on a replication-defective chimpanzee-derived adenovirus vector expressing the nucleoprotein (NP) of influenza A virus as a fusion protein with the HSV type 1 glycoprotein D, which through binding to the herpes virus entry mediator, blocks the immunoinhibitory herpes virus entry mediator B and T lymphocyte attenuator/CD160 pathways. Our results show that the vaccine expressing a fusion protein of NP and glycoprotein D induces significantly higher NP-specific CD8(+) T cell responses in young and aged mice compared with the vaccine expressing NP only.

Vaccination with Ad5 vectors expands Ad5-specific CD8 T cells without altering memory phenotype or functionality.

BACKGROUND: Adenoviral (Ad) vaccine vectors represent both a vehicle to present a novel antigen to the immune system as well as restimulation of immune responses against the Ad vector itself. To what degree Ad-specific CD8(+) T cells are restimulated by Ad vector vaccination is unclear, although such knowledge would be important as vector-specific CD8(+) T cell expansion could potentially further limit Ad vaccine efficacy beyond Ad-specific neutralizing antibody alone. METHODOLOGY/PRINCIPAL FINDINGS: Here we addressed this issue by measuring human Adenovirus serotype 5 (Ad5)-specific CD8(+) T cells in recipients of the Merck Ad5 HIV-1 vaccine vector before, during, and after vaccination by multicolor flow cytometry. Ad5-specific CD8(+) T-cells were detectable in 95% of subjects prior to vaccination, and displayed primarily an effector-type functional profile and phenotype. Peripheral blood Ad5-specific CD8(+) T-cell numbers expanded after Ad5-HIV vaccination in all subjects, but differential expansion kinetics were noted in some baseline Ad5-neutralizing antibody (Ad5 nAb) seronegative subjects compared to baseline Ad5 nAb seropositive subjects. However, in neither group did vaccination alter polyfunctionality, mucosal targeting marker expression, or memory phenotype of Ad5-specific CD8(+) T-cells. CONCLUSIONS: These data indicate that repeat Ad5-vector administration in humans expands Ad5-specific CD8(+) T-cells without overtly affecting their functional capacity or phenotypic properties. This is a secondary analysis of samples collected during the 016 trial. Results of the Merck 016 trial safety and immunogenicity have been previously published in the journal of clinical infectious diseases [1]. TRIAL REGISTRATION: ClinicalTrials.gov NCT00849680[http://www.clinicaltrials.gov/show/NCT00849680].

New insights on adenovirus as vaccine vectors.

Adenovirus (Ad) vectors were initially developed for treatment of genetic diseases. Their usefulness for permanent gene replacement was limited by their high immunogenicity, which resulted in rapid elimination of transduced cells through induction of T and B cells to antigens of Ad and the transgene product. The very trait that excluded their use for sustained treatment of genetic diseases made them highly attractive as vaccine carriers. Recently though results showed that Ad vectors based on common human serotypes, such as serotype 5, may not be ideal as vaccine carriers. A recently conducted phase 2b trial, termed STEP trial, with an AdHu5-based vaccine expressing antigens of human immunodeficiency virus 1 (HIV-1) not only showed lack of efficacy in spite of the vaccine's immunogenicity, but also suggested an increased trend for HIV acquisition in individuals that had circulating AdHu5 neutralizing antibodies prior to vaccination. Alternative serotypes from humans or nonhuman primates (NHPs), to which most humans lack pre-existing immunity, have been vectored and may circumvent the problems encountered with the use of AdHu5 vectors in humans. In summary, although Ad vectors have seen their share of setbacks in recent years, they remain viable tools for prevention or treatment of a multitude of diseases.

Adenovirus vector-induced immune responses in nonhuman primates: responses to prime boost regimens.

In the phase IIb STEP trial an HIV-1 vaccine based on adenovirus (Ad) vectors of the human serotype 5 (AdHu5) not only failed to induce protection but also increased susceptibility to HIV-1 infection in individuals with preexisting neutralizing Abs against AdHu5. The mechanisms underlying the increased HIV-1 acquisition rates have not yet been elucidated. Furthermore, it remains unclear if the lack of the vaccine's efficacy reflects a failure of the concept of T cell-mediated protection against HIV-1 or a product failure of the vaccine. Here, we compared two vaccine regimens based on sequential use of AdHu5 vectors or two different chimpanzee-derived Ad vectors in rhesus macaques that were AdHu5 seropositive or seronegative at the onset of vaccination. Our results show that heterologous booster immunizations with the chimpanzee-derived Ad vectors induced higher T and B cell responses than did repeated immunizations with the AdHu5 vector, especially in AdHu5-preexposed macaques.

Effect of preexisting immunity on an adenovirus vaccine vector: in vitro neutralization assays fail to predict inhibition by antiviral antibody in vivo.

A major obstacle to the use of adenovirus vectors derived from common human serotypes, such as human adenovirus 5 (AdHu5), is the high prevalence of virus-neutralizing antibodies in the human population. We previously constructed a variant of chimpanzee adenovirus 68 (AdC68) that maintained the fundamental properties of the carrier but was serologically distinct from AdC68 and resisted neutralization by AdC68 antibodies. In the present study, we tested whether this modified vector, termed AdCDQ, could induce transgene product-specific CD8(+) T cells in mice with preexisting neutralizing antibody to wild-type AdC68. Contrary to our expectation, the data show conclusively that antibodies that fail to neutralize the AdCDQ mutant vector in vitro nevertheless impair the vector's capacity to transduce cells and to stimulate a transgene product-specific CD8(+) T-cell response in vivo. The results thus suggest that in vitro neutralization assays may not reliably predict the effects of virus-specific antibodies on adenovirus vectors in vivo.

Potentiating vaccine immunogenicity by manipulating the HVEM/BTLA pathway and other co-stimulatory and co-inhibitory signals of the immune system.

Adaptive immune responses are tightly controlled by activating and inhibitory signals, which ensure an effective defense against pathogens while preventing detrimental reactions. Regulation is provided through sets of receptors and ligands expressed on lymphocytes and antigen-presenting cells. Expression of the regulatory molecules is up or downregulated during immune responses and some pathways provide positive or negative signals depending on the state of differentiation of the cells. Many of the ligands and receptors can engage multiple binding partners and thus have distinct effects on downstream signaling. Medicinal manipulations of such pathways are being explored to augment antigen-driven immune responses through activation of positive or blockade of negative signals. In this review we provide a brief description of the co-stimulatory and co-inhibitory receptors and ligands with focus on the pathways mediated by the herpes virus entry mediator (HVEM) and the B and T lymphocyte mediator (BTLA).

Targeting of antigen to the herpesvirus entry mediator augments primary adaptive immune responses.

Interactions between the herpesvirus entry mediator (HVEM) and the B- and T-lymphocyte attenuator (BTLA) inhibit B and T cell activation. HVEM-BTLA interactions are blocked by herpes simplex virus (HSV) glycoprotein D (gD) through binding of its N-terminal domain to the BTLA binding site of HVEM. In this study, we inserted viral antigens into the C-terminal domain of gD and expressed these antigens with plasmid or E1-deleted (replication-defective) adenovirus vectors. Viral antigens fused to gD induced T and B cell responses to the antigen that were far more potent than those elicited by the same antigen expressed without gD. The immunopotentiating effect required binding of the gD chimeric protein to HVEM. Overall, the studies demonstrate that targeting of antigen to the BTLA binding site of HVEM augments the immunogenicity of vaccines.

Recombinant adeno-associated virus vectors induce functionally impaired transgene product-specific CD8+ T cells in mice.

Recombinant adeno-associated virus (rAAV) vectors were used in human trials as carriers of vaccines for HIV-1 after encouraging preclinical results. However, the clinical trials yielded disappointing results. Here we demonstrated that in mice, rAAV vectors expressing the gene encoding HIV-1 gag stimulated gag-specific CD8(+) T cells, but these T cells failed to expand after a booster immunization with a replication-defective adenoviral (Ad) vector also expressing gag. We tested rAAV vectors of different serotypes expressing HIV-1 gag for induction of transgene product-specific CD8(+) T cells and found that the immunoinhibitory effect of rAAV priming observed with different AAV serotypes was transgene product specific, was independent of the interval between prime and boost, and extended to boosts with vaccine modalities other than Ad vectors. rAAV vector-induced CD8(+) T cells proliferated poorly, produced low levels of IFN-gamma in response to gag stimulation, and upregulated immunoinhibitory molecules. These T cells did not protect efficiently against challenge with a surrogate pathogen. Finally, we showed that the impaired proliferative capacity of the T cells was caused by persistence of the antigen-encoding rAAV vectors and could be reversed by placing the CD8(+) T cells in an antigen-free environment. Our data suggest that rAAV vectors induce functionally impaired T cells and could dampen the immune response to a natural infection.

Evaluation of experimental conditions for quantification of LT produced by human derived enterotoxigenic Escherichia coli strains

The heat-labile toxin (LT) is a key virulence-associated factor associated with the non-invasive secretory diarrhea caused by enterotoxigenic Escherichia coli (ETEC) strains either in humans or domestic animals. Several LT detection methods have been reported but quantification of the toxin produced by wild-type ETEC strains is usually performed by the GM1 ganglyoside enzyme-linked immunosorbent assay (GM1 ELISA). In this study we conducted the optimization of an alternative LT-quantification method, the antibody-capture ELISA (cELISA). Detailed analysis of the appropriate dilutions of capture and detecting LT-specific antibodies significantly improved the sensitivity of the method. Additionally, testing of different LT extraction techniques indicated that sonic disruption of the bacterial cells enhanced LT recovery yields, in contrast to the usual procedure based on addition of polymyxin B to the culture medium as well as extraction methods based on chloroform or Triton X-100. Moreover, the present data indicate that performance of the LT extraction method based on polymyxin B treatment can vary among wild ETEC strains.

A toxina termo-lábil (LT) é um fator de virulência associado à diarréia secretora não invasiva causada por linhagens de Escherichia coli enterotoxigênica (ETEC) em humanos ou animais domésticos. Diversos métodos de detecção de LT foram descritos na literatura, no entanto, a quantificação da toxina produzida por linhagens selvagens de ETEC é geralmente realizada por ensaio imunoenzimático com o gangliosídeo GM-1 (GM-1 ELISA). Neste estudo, conduzimos uma otimização experimental de um método alternativo de quantificação de LT, o ELISA de captura (cELISA). Análise detalhada de diluições apropriadas dos anticorpos LT específicos de captura e detecção melhorou significantemente a sensibilidade do método. Em adição, testes com diferentes técnicas de extração de LT indicaram que a ruptura das células por ultra-som, mas não o tratamento com polimixina B, clorofórmio ou Triton X-100, aumentou o rendimento da recuperação de LT. Além disto, os dados apresentados demonstram que o desempenho do método de extração de LT baseado no tratamento com polimixina B pode variar entre linhagens selvagens de ETEC.