Development of a VLP-Based Vaccine Displaying an xCT Extracellular Domain for the Treatment of Metastatic Breast Cancer.

Metastatic breast cancer (MBC) is the leading cause of cancer death in women due to recurrence and resistance to conventional therapies. Thus, MBC represents an important unmet clinical need for new treatments. In this paper we generated a virus-like particle (VLP)-based vaccine (AX09) to inhibit de novo metastasis formation and ultimately prolong the survival of patients with MBC. To this aim, we engineered the bacteriophage MS2 VLP to display an extracellular loop of xCT, a promising therapeutic target involved in tumor progression and metastasis formation. Elevated levels of this protein are observed in a high percentage of invasive mammary ductal tumors including triple negative breast cancer (TNBC) and correlate with poor overall survival. Moreover, xCT expression is restricted to only a few normal cell types. Here, we tested AX09 in several MBC mouse models and showed that it was well-tolerated and elicited a strong antibody response against xCT. This antibody-based response resulted in the inhibition of xCT's function in vitro and reduced metastasis formation in vivo. Thus, AX09 represents a promising novel approach for MBC, and it is currently advancing to clinical development.

Virus-Like Particles as an Immunogenic Platform for Cancer Vaccines.

Virus-like particles (VLP) spontaneously assemble from viral structural proteins. They are naturally biocompatible and non-infectious. VLP can serve as a platform for many potential vaccine epitopes, display them in a dense repeating array, and elicit antibodies against non-immunogenic substances, including tumor-associated self-antigens. Genetic or chemical conjugation facilitates the multivalent display of a homologous or heterologous epitope. Most VLP range in diameter from 25 to 100 nm and, in most cases, drain freely into the lymphatic vessels and induce antibodies with high titers and affinity without the need for additional adjuvants. VLP administration can be performed using different strategies, regimens, and doses to improve the immunogenicity of the antigen they expose on their surface. This article summarizes the features of VLP and presents them as a relevant platform technology to address not only infectious diseases but also chronic diseases and cancer.

Display of single-chain variable fragments on bacteriophage MS2 virus-like particles.

BACKGROUND: Virus-like particles (VLPs) of the RNA bacteriophage MS2 have many potential applications in biotechnology. MS2 VLPs provide a platform for peptide display and affinity selection (i.e. biopanning). They are also under investigation as vehicles for targeted drug delivery, using display of receptor-specific peptides or nucleic acid aptamers to direct their binding to specific cell-surface receptors. However, there are few molecules more suited to the precise targeting and binding of a cellular receptor than antibodies. RESULTS: Here we describe a strategy for display of four different functional single-chain variable fragments (scFvs) on the surface of the MS2 VLP. Each scFv is validated both for its presence on the surface of the VLP and for its ability to bind its cognate antigen. CONCLUSIONS: This work demonstrates the suitability of the MS2 VLP platform to display genetically fused scFvs, allowing for many potential applications of these VLPs and paving the way for future work with libraries of scFvs displayed in a similar manner on the VLP surface. These libraries can then be biopanned and novel scFv binders to targets can be readily discovered.

Epitope-Specific Anti-hCG Vaccines on a Virus Like Particle Platform.

The possibility of a contraceptive vaccine targeting human chorionic gonadotropin has long been recognized, but never fully realized. Here we describe an epitope-specific approach based on immunogenic display of hCG-derived peptides on virus-like particles of RNA bacteriophage. A number of recombinant VLPs were constructed, each displaying a different hCG-derived peptide. Some were taken from the disordered C-terminal tail of the hormone, another came from an internal loop, and yet another was an epitope mimic produced by affinity-selection on an hCG-neutralizing antibody target. Immunization of mice with some VLPs yielded antisera that bound the hormone and neutralized hCG biological activity.

A malaria vaccine candidate based on an epitope of the Plasmodium falciparum RH5 protein.

BACKGROUND: The Plasmodium falciparum protein RH5 is an adhesin molecule essential for parasite invasion of erythrocytes. Recent studies show that anti-PfRH5 sera have potent invasion-inhibiting activities, supporting the idea that the PfRH5 antigen could form the basis of a vaccine. Therefore, epitopes recognized by neutralizing anti-PfRH5 antibodies could themselves be effective vaccine immunogens if presented in a sufficiently immunogenic fashion. However, the exact regions within PfRH5 that are targets of this invasion-inhibitory activity have yet to be identified. METHODS: A battery of anti-RH5 monoclonal antibodies (mAbs) were produced and screened for their potency by inhibition of invasion assays in vitro. Using an anti-RH5 mAb that completely inhibited invasion as the selecting mAb, affinity-selection using random sequence peptide libraries displayed on virus-like particles of bacteriophage MS2 (MS2 VLPs) was performed. VLPs were sequenced to identify the specific peptide epitopes they encoded and used to raise specific antisera that was in turn tested for inhibition of invasion. RESULTS: Three anti-RH5 monoclonals (0.1 mg/mL) were able to inhibit invasion in vitro by >95%. Affinity-selection with one of these mAbs yielded a VLP which yielded a peptide whose sequence is identical to a portion of PfRH5 itself. The VLP displaying the peptide binds strongly to the antibody, and in immunized animals elicits an anti-PfRH5 antibody response. The resulting antisera against the specific VLP inhibit parasite invasion of erythrocytes more than 90% in vitro. CONCLUSIONS: Here, data is presented from an anti-PfRH5 mAb that completely inhibits erythrocyte invasion by parasites in vitro, one of the few anti-malarial monoclonal antibodies reported to date that completely inhibits invasion with such potency, adding to other studies that highlight the potential of PfRH5 as a vaccine antigen. The specific neutralization sensitive epitope within RH5 has been identified, and antibodies against this epitope also elicit high anti-invasion activity, suggesting this epitope could form the basis of an effective vaccine against malaria.

Cell-specific delivery of diverse cargos by bacteriophage MS2 virus-like particles.

Virus-like particles (VLPs) of bacteriophage MS2 possess numerous features that make them well-suited for use in targeted delivery of therapeutic and imaging agents. MS2 VLPs can be rapidly produced in large quantities using in vivo or in vitro synthesis techniques. Their capsids can be modified in precise locations via genetic insertion or chemical conjugation, facilitating the multivalent display of targeting ligands. MS2 VLPs also self-assemble in the presence of nucleic acids to specifically encapsidate siRNA and RNA-modified cargos. Here we report the use of MS2 VLPs to selectively deliver nanoparticles, chemotherapeutic drugs, siRNA cocktails, and protein toxins to human hepatocellular carcinoma (HCC). MS2 VLPs modified with a peptide (SP94) that binds HCC exhibit a 10(4)-fold higher avidity for HCC than for hepatocytes, endothelial cells, monocytes, or lymphocytes and can deliver high concentrations of encapsidated cargo to the cytosol of HCC cells. SP94-targeted VLPs loaded with doxorubicin, cisplatin, and 5-fluorouracil selectively kill the HCC cell line, Hep3B, at drug concentrations <1 nM, while SP94-targeted VLPs that encapsidate a siRNA cocktail, which silences expression of cyclin family members, induce growth arrest and apoptosis of Hep3B at siRNA concentrations <150 pM. Impressively, MS2 VLPs, when loaded with ricin toxin A-chain (RTA) and modified to codisplay the SP94 targeting peptide and a histidine-rich fusogenic peptide (H5WYG) that promotes endosomal escape, kill virtually the entire population of Hep3B cells at an RTA concentration of 100 fM without affecting the viability of control cells. Our results demonstrate that MS2 VLPs, because of their tolerance of multivalent peptide display and their ability to specifically encapsidate a variety of chemically disparate cargos, induce selective cytotoxicity of cancer in vitro and represent a significant improvement in the characteristics of VLP-based delivery systems.

Thermal stability of RNA phage virus-like particles displaying foreign peptides.

BACKGROUND: To be useful for genetic display of foreign peptides a viral coat protein must tolerate peptide insertions without major disruption of subunit folding and capsid assembly. The folding of the coat protein of RNA phage MS2 does not normally tolerate insertions in its AB-loop, but an engineered single-chain dimer readily accepts them as long as they are restricted to one of its two halves. RESULTS: Here we characterize the effects of peptide insertions on the thermal stabilities of MS2 virus-like particles (VLPs) displaying a variety of different peptides in one AB-loop of the coat protein single-chain dimer. These particles typically denature at temperatures around 5-10°C lower than unmodified VLPs. Even so, they are generally stable up to about 50°C. VLPs of the related RNA phage PP7 are cross-linked with intersubunit disulfide bonds and are therefore significantly more stable. An AB-loop insertion also reduces the stability of PP7 VLPs, but they only begin to denature above about 70°C. CONCLUSIONS: VLPs assembled from MS2 single-chain dimer coat proteins with peptide insertions in one of their AB-loops are somewhat less stable than the wild-type particle, but still resist heating up to about 50°C. Because they possess disulfide cross-links, PP7-derived VLPs provide an alternate platform with even higher stability.

Peptide epitope identification by affinity selection on bacteriophage MS2 virus-like particles.

Filamentous phages are now the most widely used vehicles for phage display and provide efficient means for epitope identification. However, the peptides they display are not very immunogenic because they normally fail to present foreign epitopes at the very high densities required for efficient B-cell activation. Meanwhile, systems based on virus-like particles (VLPs) permit the engineered high-density display of specific epitopes but are incapable of peptide library display and affinity selection. We developed a new peptide display platform based on VLPs of the RNA bacteriophage MS2. It combines the high immunogenicity of MS2 VLPs with the affinity selection capabilities of other phage display systems. Here, we describe plasmid vectors that facilitate the construction of high-complexity random sequence peptide libraries on MS2 VLPs and that allow control of the stringency of affinity selection through the manipulation of display valency. We used the system to identify epitopes for several previously characterized monoclonal antibody targets and showed that the VLPs thus obtained elicit antibodies in mice whose activities mimic those of the selecting antibodies.

Corticosterone evaluation in Wistar rats infected with the Y strain of Trypanosoma cruzi during the chronic phase.

Understanding the mechanisms responsible for mediating the effects of stress on Trypanosoma cruzi infection is crucial for determining the full impact of stress on Chagas' disease and for devising effective interventions. Dehydroepiandrosterone (DHEA), a steroid hormone synthesized from pregnenolone, is secreted by the adrenal cortex in response to stress. Although its physiologic role has not been fully defined, DHEA has been shown to modulate immune function. In the present study, we evaluated the levels of corticosterone and the ability of T. cruzi infection to modulate the expression of Th2 cytokines in Wistar rats with chronic Chagas' disease submitted to repetitive stress. The animals submitted to stress displayed enhanced levels of corticosterone as compared to control counterparts. Stress and infection triggered the most elevated concentrations of corticosterone. DHEA significantly reduced corticosterone levels for infected and stressed animals with DHEA. The infected animals displayed enhanced levels of IL-10 and IL-4 as compared to control ones. Stress combined with infection triggered the higher levels of IL-10 and IL-4. DHEA alone and combined with infection and stress significantly increased IL-10 and IL-4 levels. Then, this study might provide additional clues about factors that regulate some of the immunoregulatory aspects of T. cruzi infection and might offer new opportunities for therapeutic interventions.

Immunogenic display of diverse peptides, including a broadly cross-type neutralizing human papillomavirus L2 epitope, on virus-like particles of the RNA bacteriophage PP7.

The immunogenicity of an antigen can be dramatically increased by displaying it in a dense, multivalent context, such as on the surface of a virus or virus-like particle (VLP). Here we describe a highly versatile VLP platform for peptide display based on VLPs of the RNA bacteriophage PP7. We show that this platform can be used for the engineered display of specific peptide sequences as well as for the construction of random peptide libraries. Peptides representing the FLAG epitope, the V3 loop of HIV gp120, and a broadly cross-type neutralizing epitope from L2, the minor capsid protein of Human Papillomavirus type 16 (HPV16), were inserted into an exposed surface loop of a form of PP7 coat protein in which the two identical polypeptides of coat were fused together to form a single-chain dimer. The recombinant proteins assembled into VLPs, displayed these peptides on their surfaces, and induced high-titer antibody responses. The single-chain dimer was also highly tolerant of random 6-, 8-, and 10-amino acid insertions. PP7 VLPs displaying the HPV16 L2 epitope generated robust anti-HPV16 L2 serum antibodies after intramuscular injection that protected mice from genital infection with HPV16 pseudovirus as well as a heterologous HPV pseudovirus type, HPV45. Thus, PP7 VLPs are well-suited for the display of a wide diversity of peptides in a highly immunogenic format.

Growth hormones therapy in immune response against Trypanosoma cruzi.

Growth hormone (GH) is an important hypophyseal hormone that is primarily involved in body growth and metabolism. In mammals, control of Trypanosoma cruzi parasitism during the acute phase of infection is considered to be critically dependent on direct macrophage activation by cytokines. To explore the possibility that GH might be effective in the treatment of Chagas' disease, we investigated its effects on the course of T. cruzi infection in rats, focusing our analyses on its influences on parasitemia, NO, TNF-alpha and IFN-gamma concentration and on histopathological alterations and parasite burden in heart tissue. T. cruzi-infected male Wistar rats were intraperitoneally treated with 5 ng/10 g body weight/day of GH. Animals treated with GH showed a significant reduction in the number of blood trypomastigotes during the acute phase of infection compared with untreated animals (P<0.05). For all experimental days (7, 14 and 21 post infection) of the acute phase, infected and GH treated animals reached higher concentrations of TNF-alpha, IFN-gamma and nitric oxide as compared to untreated and infected counterparts (P<0.05) Histopathological observations of heart tissue revealed that GH administration also resulted in fewer and smaller amastigote burdens, and less inflammatory infiltrate and tissue disorganization, indicating a reduced parasitism of this tissue. These results show that GH can be considered as an immunomodulator substance for controlling parasite replication and combined with the current drug used may represent in the future a new therapeutic tool to reduce the harmful effects of Chagas' disease.

Trypanosoma cruzi: dehydroepiandrosterone (DHEA) and immune response during the chronic phase of the experimental Chagas' disease.

Dehydroepiandrosterone (DHEA) has long been considered as a precursor for many steroid hormones. It also enhances the immune responses against a wide range of viral, bacterial, and parasitic pathogens. The aims of this work were to evaluate the influences of exogenous DHEA treatment on Wistar rats infected with the Y strain of Trypanosoma cruzi during the acute and its influence on the chronic phase of infection. Animals were subcutaneous treated with 40 mg/kg body weight/day of DHEA. DHEA treatment promoted increased lymphoproliferative responses as well as enhanced concentrations of NO and IL-12. So, we point in the direction that our results validate the utility of the use of DHEA as an alternative therapy candidate against T. cruzi.

Effects of repetitive stress during the acute phase of Trypanosoma cruzi infection on chronic Chagas' disease in rats.

The effect of repetitive stress during acute infection with Trypanosoma cruzi (T. cruzi) on the chronic phase of ensuing Chagas' disease was the focus of this investigation. The aim of this study was to evaluate in Wistar rats the influence of repetitive stress during the acute phase of infection (7 days) with the Y strain of T. cruzi on the chronic phase of the infection (at 180 days). Exposure to ether vapor for 1 min twice a day was used as a stressor. Repetitive stress enhanced the number of circulating parasites and cardiac tissue disorganization, from a moderate to a severe diffuse mononuclear inflammatory process and the presence of amastigote burden in the cardiac fibers. Immunological parameters revealed that repetitive stress triggered a reduced concanavalin A induced splenocyte proliferation in vitro with major effects on the late chronic phase. Serum interleukin-12 concentration decreased in both stressed and infected rats in the early phase of infection although it was higher on 180 days post-infection. These results suggest that repetitive stress can markedly impair the host's immune system and enhance the pathological process during the chronic phase of Chagas' disease.

Trypanosoma cruzi: effects of adrenalectomy during the acute phase of experimental infection.

Glucocorticoid hormones have been implicated as an important modulator of Trypanosoma cruzi pathogenesis. Since adrenal steroid hormones play a fundamental role in modulating the immune response, we hypothesized that adrenalectomy affect the course of the experimental T. cruzi infection. This study was undertaken to determine the effects of adrenalectomy during the acute phase of T. cruzi infection. Blood and tissue parasitism, macrophages, nitric oxide (NO) production and IFN-gamma were evaluated in male Wistar rats infected with the Y strain of T. cruzi. Our results show that adrenalectomized rats displayed increased number of blood and heart parasites accompanied by decreases in the total number of peritoneal macrophages and IFN-gamma when compared to controls. Adrenalectomy also reduced the levels of NO released from peritoneal macrophages of infected animals. These results suggest that adrenal corticosteroid insufficiency due to adrenalectomy could be considered an important factor during development of acute phases of experimental Chagas' disease, enhancing pathogenesis through disturbance of the host's immune system.

Immunogenic display of diverse peptides on virus-like particles of RNA phage MS2.

The high level of immunogenicity of peptides displayed in dense repetitive arrays on virus-like particles makes recombinant VLPs promising vaccine carriers. Here, we describe a platform for vaccine development based on the VLPs of RNA bacteriophage MS2. It serves for the engineered display of specific peptide sequences, but will also allow the construction of random peptide libraries from which specific binding activities can be recovered by affinity selection. Peptides representing the V3 loop of HIV gp120 and the ECL2 loop of the HIV coreceptor, CCR5, were inserted into a surface loop of MS2 coat protein. Both insertions disrupted coat VLP assembly, apparently by interfering with protein folding, but these defects were suppressed efficiently by genetically fusing coat protein's two identical polypeptides into a single-chain dimer. The resulting VLPs displayed the V3 and ECL2 peptides on their surfaces where they showed the potent immunogenicity that is the hallmark of VLP-displayed antigens. Experiments with random-sequence peptide libraries show the single-chain dimer to be highly tolerant of six, eight and ten amino acid insertions. MS2 VLPs support the display of a wide diversity of peptides in a highly immunogenic format, and they encapsidate the mRNAs that direct their synthesis, thus establishing the genotype/phenotype linkage necessary for recovery of affinity-selected sequences. The single-chain MS2 VLP therefore unites in a single structural platform the selective power of phage display with the high immunogenicity of VLPs.

Stability and assembly in vitro of bacteriophage PP7 virus-like particles.

BACKGROUND: The stability of a virus-like particle (VLP) is an important consideration for its use in nanobiotechnology. The icosahedral capsid of the RNA bacteriophage PP7 is cross-linked by disulfide bonds between coat protein dimers at its 5-fold and quasi-6-fold symmetry axes. This work determined the effects of these disulfides on the VLP's thermal stability. RESULTS: Measurements of the thermal denaturation behavior of PP7 VLPs in the presence and absence of a reducing agent show that disulfide cross-links substantially stabilize them against thermal denaturation. Although dimers in the capsid are linked to one another by disulfides, the two subunits of dimers themselves are held together only by non-covalent interactions. In an effort to confer even greater stability a new cross-link was introduced by genetically fusing two coat protein monomers, thus producing a "single-chain dimer" that assembles normally into a completely cross-linked VLP. However, subunit fusion failed to increase the thermal stability of the particles, even though it stabilized the isolated dimer. As a step toward gaining control of the internal composition of the capsid, conditions that promote the assembly of PP7 coat protein dimers into virus-like particles in vitro were established. CONCLUSION: The presence of inter-dimer disulfide bonds greatly stabilizes the PP7 virus-like particle against thermal denaturation. Covalently cross-linking the subunits of the dimers themselves by genetically fusing them through a dipeptide linker sequence, offers no further stabilization of the VLP, although it does stabilize the dimer. PP7 capsids readily assemble in vitro in a reaction that requires RNA.

Trypanosoma cruzi: plasma corticosterone after repetitive stress during the acute phase of infection.

An increased level of plasma corticosterone is one manifestation of severe environmental or physiologic stress. The stress response mediated by the hypothalamic-pituitary-adrenal axis is already known to suppress immunoglobulin production and to impair immune function, but there are few studies relating stress and plasma corticosterone to the outcome of Trypanosoma cruzi infection. In this study, male Wistar rats were infected with the Y strain of T. cruzi and then subjected to repetitive stress by exposure to ether vapor for 1min twice a day during the acute phase of infection. Stressed animals showed decreased lytic antibody activity and lowered levels of peritoneal macrophages. Despite an increase in the weight of the spleen, histological analyses demonstrated tissue alterations, the presence of amastigote nests, and a complete absence of activated lymphoid follicles. These results suggest that stress-induced increases in plasma corticosterone can suppress the immune response and worsen tissue injury during the acute phase of T. cruzi infection.

Trypanosoma cruzi: effects of repetitive stress during the development of experimental infection.

Activation of the hypothalamus-pituitary-adrenal axis plays a major role in the suppression of the immune system. We have investigated the effects of repetitive stress on Wistar rats infected with the Y strain of Trypanosoma cruzi and a control group that underwent stressor stimuli by exposure to ether vapor for one minute twice a day. Repetitive stress resulted in an elevated number of circulating parasites accompanies by deep tissue disorganization, and cardiac histopathological alterations. The infected and stressed group displayed a decrease in body weight, and an increased parasite burden in heart tissue, and adrenal glands. Histological analysis of the heart also showed a moderate to severe diffused mononuclear inflammatory process. These results suggest that repetitive stress could be considered an important factor during development of experimental Chagas' disease, enhancing pathogenesis through disturbance of the host's immune system.

Fetal alcohol exposure alters neurosteroid levels in the developing rat brain.

Neurosteroids are modulators of neuronal function that may play important roles in brain maturation. We determined whether chronic prenatal ethanol exposure altered neurosteroid levels in the developing brain. Rat dams were exposed to: (i) a 5% ethanol-containing liquid diet that produces peak maternal blood alcohol levels near the legal intoxication limit (approximately 0.08 g/dL); (ii) an isocaloric liquid diet containing maltose-dextrin instead of ethanol with pair-feeding; (iii) rat chow ad libitum. Neurosteroid levels were assessed in offspring brains using radioimmunoassay or gas chromatography-mass spectrometry techniques. A prenatal ethanol exposure-induced increase in pregnenolone sulfate levels, but not dehydroepiandrosterone sulfate levels, was evident at the earliest time point studied (embryonic day 14). This effect lasted until post-natal day 5. Levels of other neurosteroids were assessed at embryonic day 20; pregnenolone levels, but not allopregnanolone levels, were elevated. Pregnenolone sulfate levels were not altered in the maternal brain. Neither pregnenolone nor pregnenolone sulfate levels were significantly altered in the fetal liver, placenta and maternal blood, indicating that the effect of ethanol is not secondary to accumulation of peripherally-produced steroids. Fetal ethanol exposure has been shown to decrease both cellular and behavioral responsiveness to neurosteroids, and our findings provide a plausible explanation for this effect.

Consequências do estresse na atividade do eixo hipotálamo-hipófise-adrenal e no envelhecimento / Consequences of stress in hypothalamic-pituitary-adrenal axis activity and agind

o estresse não é um fenômeno da vida contemporânea, mas inato ao organismo, uma vez que o primeiro ser já deveria sofrer dificuldades para manter seu equilíbrio. O assunto é muito discutido atualmente em decorrência das consequências negativas do estresse na vida diária das pessoas, tais como cansaço, irritabilidade, labilidade emocional, fragilidade imunológica, gastrite, asma. Contudo, há também os efeitos benéficos, já que o estresse é essencial à vida. O preocesso de envelhecimento sempre foi motivo de preocupação para a medicina, entretanto, das centenas de trabalhos publicados sobre as mudanças na idade avançada, não se sabe quais os mecanismos exatos que levam à senescência. todavia, nos últimos trinta anos houve progressos notáveis, sobretudo devido a trabalhos experimentais que demonstram os papéis significativos de agentes externos e internos no processo de envelhecimento. Este trabalho teve por objetivo discutir como o organismo reage aos estímulos estressores, enfatizando o eixo hipotálamo-hipófise-adrenal, as mudanças fisiológicas e bioqu´´imicas que ocorrem tanto no jovem como no idoso, o momento em que estas mudanças passam a ser prejudiciais e a sua associação com as doenças do envelhecimento. É também destacada a participação do estresse no processo normal de snescência