Stability in human serum and plasma of the HIV peptide drug candidate CIGB-210 and improved variants.

The peptide CIGB-210 inhibits HIV replication, inducing a rearrangement of vimentin intermediate filaments. The assessment of the in vitro serum and plasma stability of this peptide is important to develop an optimal pharmacological formulation. A half-life of 17.68 ± 0.59 min was calculated for CIGB-210 in human serum by reverse-phase high-performance liquid chromatography (HPLC) and mass spectrometry (MS). Eight metabolites of CIGB-210 were identified with this methodology, all of them lacking the N-terminal moiety. A previously developed CIGB-210 in-house competitive ELISA was used to compare the stability of CIGB-210 derivatives containing either D-amino acids, acetylation at the N-terminus, or both modifications. The half-life of CIGB-210 in serum was five times higher when measured by ELISA than by HPLC/MS, and twice higher in plasma as compared to serum. The substitution of D-asparagine on position 6 doubled the half-life, while D-amino acids on positions 8 and 9 did not improve the stability. The acetylation of the N-terminus resulted in a 24-fold more stable peptide in plasma. The positive effect of N-terminal acetylation on CIGB-210 serum stability was confirmed by the HPLC/MS method, as the half-life of the peptide was not reached after 2 h of incubation, which represents more than a 6.8-fold increase in the half-life with respect to the original peptide.

A competitive ELISA for the quantitative determination of the novel anti-HIV drug candidate CIGB-210 in biological fluids.

The synthetic peptide CIGB-210 is a promising anti-HIV drug candidate shown to inhibit HIV replication in MT4 cells at the nanomolar range by triggering the rearrangement of vimentin intermediate filaments. Sensitive and specific analytical methods are required for pharmacological studies of CIBG-210 in animals. In this study, we describe the development of a competitive ELISA for the quantitative determination of CIGB-210 using an anti-CIGB-210 hyperimmune serum. After optimization of all the steps, the assay exhibited a dynamic range from 11.87 to 0.0095 µg/mL. The intra-assay coefficient of variation (CV) was lower than or close to 5% for all the six concentrations of the calibrator, and the inter-assay CV was below 10% in five out of the six concentrations tested. No interference of either murine or human plasma was observed. The analyte was stable in plasma after five freeze-thaw cycles, while the hyperimmune serum maintained its binding capacity after 10 freeze-thaw cycles. Furthermore, the ELISA was able to detect the two main metabolites of CIGB-210, although with a tenfold decrease in sensitivity. Our results demonstrate the utility and feasibility of this analytical method for pharmacological experiments in animals as humans.

An improved method for purification and refolding of recombinant HIV Vif expressed in Escherichia coli.

Virion infectivity factor (Vif) is a 23 kDa protein that protects HIV-1 from deamination of its proviral DNA by APOBEC3G. The active form of Vif is a multimer that interacts simultaneously with CBF-beta, the elongin B and C subunits, Cullin 5, and APOBEC3G to form a ubiquitin ligase complex targeting the latter for degradation. Vif clearly represents an attractive target for developing novel antiviral drugs for the therapy of HIV/AIDS, and this goal requires a source of well folded, readily available protein. For that purpose, we have cloned Vif in the pET28a expression vector, expressing the resulting His-tagged recombinant protein in the BL21(DE3) Escherichia coli strain. After lysis, Vif was solubilized from the insoluble fraction with 6 M guanidinium chloride and purified by denaturing immobilized-metal affinity chromatography, refolding the protein afterwards by dialysis. The use of 2-(N-morpholino)ethanesulfonic acid buffer at pH 6.2 and the presence of EDTA improved Vif refolding yields by reducing the formation of insoluble aggregates. The purified protein was bound by two monoclonal antibodies against sequential and conformational epitopes located at the C and N terminus, respectively.

Preliminary safety assessment of CIGB-210, an investigational peptide for HIV infection.

Current human immunodeficiency virus treatments need to be periodically administered lifelong. In this study we assess the effect of repeated doses of an anti-HIV peptide drug candidate in C57BL6 strain. Two schemes of up to 15 administrations and one of 30, daily dosing for 5 days per week, all by the subcutaneous route were evaluated. Different dose concentrations of the peptide were assayed. CIGB-210 treated animals showed no symptoms or abnormal behavior as compared with placebo. All the animals gained weight during the study. Macroscopic evaluation showed no alterations in any of the organs studied. Microscopic analysis of the tissues did not show morphological changes in thymus, stomach, small and large intestines, kidney, brain, or cerebellum. The proliferative response of splenocytes and their capacity to secrete gamma interferon were not compromised by the repeated administration of CIGB-210. There were not statistically significant differences for any of the parameters evaluated during the study among treated and non-treated groups. We can conclude that CIGB-210 is well tolerated in C57BL6 mice in the dose concentration range explored and merits subsequent toxicological studies.

E2-CD154 vaccine candidate is safe and immunogenic in pregnant sows, and the maternal derived neutralizing antibodies protect piglets from classical swine fever virus challenge.

E2-CD154 subunit vaccine candidate is safe and protects swine from Classical Swine Fever (CSF). However, its safety and immunogenicity in pregnant sows, and the capacity of maternal derived neutralizing antibodies (MDNA) to protect the offspring is yet to be demonstrated. The aim of this study was to evaluate the safety and immunogenicity of E2-CD154 in pregnant sows, and the capacity of MDNA to protect the offspring. Seventeen pregnant sows were vaccinated twice with E2-CD154 in either the first or the second third of pregnancy. Pregnancy and litter parameters were compared with a control group of non-vaccinated sows. Neutralizing antibodies (NAb) were monitored. The time course of MDNA was assessed in a group of six piglets born to an E2-CD154 immunized sow, and the animals were challenged with CSFV at day 63 after birth. No local or systemic adverse effects were found. Neither abortions, nor congenital malformations, nor stillbirths were observed. All sows develop high NAb titers after the first immunization. Piglets born to an E2-CD154 vaccinated sow still showed MDNA titers of 1:100 at day 63 after birth. Five animals were negative for virus isolation after challenge, and showed neither signs of CSF, nor macroscopic lesions in the organs. The other piglet was positive for CSFV isolation, and macroscopic lesions were observed in the spleen, although no clinical signs of CSF other than fever were detected. E2-CD154 vaccine candidate was safe and immunogenic in pregnant sows, and the passive immunity transmitted to the offspring was still protective by day 63 after birth.

Novel chimeric E2CD154 subunit vaccine is safe and confers long lasting protection against classical swine fever virus.

E2CD154 is a vaccine candidate against classical swine fever (CSF) based on a chimeric protein composed of the E2 glycoprotein fused to porcine CD154 antigen, and formulated in the oil adjuvant Montanide™ ISA 50 V2. This vaccine confers early protection in pigs and prevents vertical transmission in pregnant sows. The objectives of this study were to assess the safety of this immunogen in piglets, to compare several doses of antigen in the formulation, and to study the duration of the immunity provided by this vaccine for up to 9 months. Three trials were conducted by immunizing pigs with a two-dose regime of the vaccine. Challenge experiments were carried out with the highly pathogenic Margarita strain. No local or systemic adverse effects were documented, and neither macroscopic nor microscopic pathological findings were observed in the vaccinated animals. The three antigen doses explored were safe and induced CSF protective neutralizing antibodies. The dose of 50 µg was selected for further development because it provided the best clinical and virological protection. Finally, this protective immunity was sustained for at least 9 months. This study demonstrates that E2CD154 vaccine is safe; defines a vaccine dose of 50 µg antigen, and evidences the capacity of this vaccine to confer long term protection from CSFV infection for up to 9 months post- vaccination. These findings complement previous data on the evaluation of this vaccine candidate, and suggest that E2CD154 is a promising alternative to modified live vaccines in CSF endemic areas.

Immunogenicity of E2CD154 Subunit Vaccine Candidate against Classical Swine Fever in Piglets with Different Levels of Maternally Derived Antibodies.

E2CD154 is a novel subunit vaccine candidate against classical swine fever virus (CSFV). It contains the E2 envelope protein from CSFV fused to the porcine CD154 molecule formulated in the oil adjuvant MontanideTM ISA50 V2. Previous works evidenced the safety and immunogenicity of this candidate. Here, two other important parameters related to vaccine efficacy were assessed. First, the existence of high maternally derived antibody (MDA) titers in piglets born to sows vaccinated with E2CD154 was demonstrated. These MDA titers remained above 1:200 during the first seven weeks of life. To assess whether the titers interfere with active vaccination, 79 piglets from sows immunized with either E2CD154 or a modified live vaccine were vaccinated with E2CD154 following a 0-21-day biphasic schedule. Animals immunized at either 15, 21, or 33 days of age responded to vaccination by eliciting protective neutralizing antibody (NAb) titers higher than 1:600, with a geometric mean of 1:4335, one week after the booster. Those protective levels of NAb were sustained up to six months of age. No vaccination-related adverse effects were described. As a conclusion, E2CD154 is able to induce protective NAb in piglets with different MDA levels and at different days of age.

Induction of a strong HIV-specific CD8+ T cell response in mice using a fowlpox virus vector expressing an HIV-1 multi-CTL-epitope polypeptide.

Recombinant avipoxvirus vectors are attractive candidates for use in vaccination strategies for infections such as human immunodeficiency virus type 1 (HIV-1), where induction of a CD8+ T cell response is thought to be an important component of protective immunity. Here, we report the expression of a multiepitope polypeptide (TAB9) composed of the central 15 amino acids of the V3 loop from six different isolates of HIV-1 in a fowlpox virus (FWPV) vector, and the use of this vector (FPTAB9LZ) to induce strong HIV-specific CD8+ T cell responses in mice. In animals immunized twice intravenously with FPTAB9LZ, almost 2% of the CD8+ T cells in the spleen were shown to produce IFN-gamma in response to stimulation with HIV-1 peptides 1 week after the second immunization. The most dominant response was to the HIV-1 IIIB peptide. A strong HIV-specific response was also induced by intraperitoneal immunization of mice with FPTAB9LZ, whilst subcutaneous immunization elicited a weaker response. Intraperitoneal immunization with FPTAB9LZ was also shown to provide protection against challenge with a recombinant vaccinia virus expressing antigens, including those in TAB9. These results confirm the potential of FWPV vectors for use in HIV vaccination strategies.

A heterogeneous enzymatic assay for quantification of Plasmepsin II activity and the evaluation of its inhibitors.

The emergence and worldwide spreading of Plasmodium falciparum strains that shown to be resistant to traditional drugs is considered a very serious health problem, given the high mortality and morbidity rate of Malaria. In the search for new drugs against this parasite, Hb hydrolyzing enzymes, such as Plasmepsin II (Plm II), have been classified as very promising targets for therapeutic attacks. In this work, it is developed a cheap and high-throughput heterogeneous enzymatic assay for measuring Plasmepsin II activity in order to use it as a tool in the discovery of new inhibitors of this enzyme. In this assay, Plasmepsin II acts upon a solid-phase bound synthetic peptide (DU2) whose sequence comprises the cleavage site F(33)-L(34) present in Hb alpha-chain. The peptide surface density is quantified by means of a classical ELISA-based procedure. In order to estimate the kinetic constants of the system and to quantify both, enzymatic and inhibitory activity, it was used a model for the kinetics of enzyme quasi-saturable systems previously developed by our group, that fitted very well to the experimental data. It was used Pepstatin as a model inhibitor of Plasmepsin II and the resulting dose-response relation agreed with the expected behavior for the Pepstatin-Plasmepsin II pair under the employed experimental conditions.

Effect of promoters on cellular immune response induced by recombinant fowlpox virus expressing multi-epitope polypeptides from HIV-1.

HIV stimulates strong immune CD8(+) cytotoxic T lymphocytes (CTL) response in infected people, despite causing an immunodeficiency. It has been demonstrated that this response could be very important for the control of the virus. We have shown previously that a recombinant fowlpox virus (rFWPV), expressing the multi-epitope polypeptide (MEP) from HIV-1 TAB9, induces strong and protective Th1 and CTL responses in Balb/c mice. Here, we have studied the CTL response against MEPs TAB9 and CR3 after immunizing with rFWPVs, where these genes are under the control of a strong synthetic early/late promoter or the 7.5 kDa promoter from vaccinia virus. TAB9 expression was increased by more than 9-fold using the strong promoter, which was translated into a two times increase in CTL response. The overall expression of CR3 was already ten times higher when compared with TAB9 with the 7.5 kDa promoter, but the use of a stronger promoter showed no effect either on the expression or CTL response. Moreover, rFWPV expressing TAB9 induced a stronger CTL response than those expressing CR3, measured as the number of interferon- gamma -secreting splenocytes, in spite of its lower antigen expression levels. These results suggest that the capacity of a stronger promoter to increase the MEP expression and/or CTL response against their epitopes is highly dependent on the nature of the polypeptide used.

Modified-vaccinia-virus-Ankara (MVA) priming and fowlpox-virus booster elicit a stronger CD8+ T-cell response in mice against an HIV-1 epitope than does a DNA/poxvirus prime-booster approach.

A prime-boost strategy combining FWPV (fowlpox virus) and the MVA (modified vaccinia virus Ankara), both expressing HIV-1 multi-V3 epitope polypeptides, was compared with a DNA-based Semliki Forest virus replicon/poxvirus approach for the induction of a CD8(+) T-cell response. Priming mice with recombinant MVA and boosting with recombinant FWPV, and not in the reverse order, increased the number of specific interferon-gamma-secreting cells in relation to the homologous combinations. Moreover, the improvement of the CD8(+) T-cell response with this combination was remarkably higher than that obtained by priming with a DNA vector containing a Semliki Forest virus replicon expressing the multi-epitope polypeptide and boosting either with recombinant MVA or FWPV. These results open a new and attractive alternative for vaccine preparation against HIV-1 using different immunogens.

Enhanced cell-mediated IFN-gamma-secreting activity against the HIV-1IIIB V3 peptide of the TAB9 multiepitope after DNA vaccine backbone engineering.

The SV40t polyadenylation and splicing signals of the pAEC plasmid vectors were replaced by synthetic intron and synthetic rabbit beta globin-based termination/polyadenylation sequences, and 5, 10, and 20 copies of the 5'-AACGTT-3' CpG motif were inserted. Balb/c mice were immunized by intramuscular injection of 200 microg of each plasmid, coding for the HIV-1 multiepitope TAB9, under the control of the human cytomegalovirus promoter. After three doses of DNA, a fourth boost with plasmid DNA or a TAB9-expressing recombinant fowlpox virus rFPTAB9LZ was administered. ELISA and ELISPOT assays were conducted for antibody and IFN-gamma-secreting cell-mediated responses' evaluation against the whole TAB9 and the TAB9's IIIB V3 peptide, respectively. Serum IgG antibodies were not detected. Effector IFN-gamma-secreting responses were only detected on the animals receiving the new set of DNA constructs, alone or in combination with a recombinant virus boost, with or without in vitro re-stimulation. The response was dependent on the new transcriptional unit and influenced by the number of CpG motifs. We showed that plasmid backbone optimization based on these two factors could enhance the response against a multiepitope-based DNA vaccine. A new family of plasmid vectors is also available for evaluation with desired antigens.

The HIV-1 chimeric protein CR3 expressed by poxviral vectors induces a diverse CD8+ T cell response in mice and is antigenic for PBMCs from HIV+ patients.

Recombinant avipoxvirus vectors are attractive for vaccination against human immunodeficiency virus type 1 (HIV-1), where induction of a cytotoxic CD8(+) T cell (CTL) response seems to be an important component of protective immunity. We expressed the chimeric protein CR3, composed by CTL epitopes rich regions from, RT, Gag and Nef and conserved Th cell epitopes from gp120, gp41 and Vpr of HIV-1 in a fowlpox virus (FWPV) vector (FPCR3), and used this vector to induce HIV-specific CTL responses in mice. Mice immunised twice intraperitoneally with FPCR3, developed a CD8(+) T cell response measured as production of IFN-gamma by splenocytes in response to stimulation with P815 cells infected with recombinant vaccinia viruses (rVV) expressing CR3, Gag and Nef. The number of IFN-gamma secreting cells was markedly higher when a P815 cell line constitutively expressing CR3 was used as target cells for Enzyme-linked-immunospot (ELISPOT). CR3 epitopes were also specifically recognised by human PBMCs from three HIV(+) patients with different haplotypes. These results confirm the potential of FWPV vectors expressing these novel HIV-1 chimeric proteins to induce a simultaneous CD8(+) T cell response against conserved viral targets and early expressed regulatory proteins.

An immunoenzymatic solid-phase assay for quantitative determination of HIV-1 protease activity.

A novel immunoenzymatic procedure for the quantitative determination of HIV protease activity is provided. An N-terminal biotinylated peptide (DU1) that comprises an HIV-1 protease (HIV-PR) cleavage sequence was bound to streptavidin-coated microtiter plates. The bound peptide can be quantified by an immunoenzymatic procedure (enzyme-linked immunosorbent assay, ELISA) that includes a monoclonal antibody (Mab 332) against the peptide (DU1) C-terminal. The incubation of the bound peptide with HIV-PR in solution resulted in a signal decrement, as the peptide was hydrolyzed and the released C-terminal segment washed away. An equation that relates the amount of added enzyme to the kinetics of the reaction was written in order to describe this heterogeneous enzyme-quasi-saturable system. This equation allows quantitative determination of protease activity, a feature widely underrated in previous similar assays. The assay also allows evaluation of the inhibitory activity of HIV-PR inhibitors. Due to the intrinsic advantages of the ELISA format, this method could be used in high-throughput screening of HIV protease inhibitors. The assay can be extended to other proteolytic enzymes.