Searching Epitope-Based Vaccines Using Bioinformatics Studies.

Epitope-based vaccines is one of the most recent methodologies applied in bioinformatics studies. This strategy consists of identifying regions of the protein (peptides or epitopes) which show antigen properties capable of stimulating the immune system against proteins from virus, bacteria, fungi, etc. This chapter describes a general procedure to identify epitopes to be used as epitope vaccine using bioinformatics methods including primary protein sequence analyses, epitope predictor, docking, and molecular dynamics simulations for the selection of T- and B-cell epitopes.

Bioinformatics design and experimental validation of influenza A virus multi-epitopes that induce neutralizing antibodies.

Pandemics caused by influenza A virus (IAV) are responsible for the deaths of millions of humans around the world. One of these pandemics occurred in Mexico in 2009. Despite the impact of IAV on human health, there is no effective vaccine. Gene mutations and translocation of genome segments of different IAV subtypes infecting a single host cell make the development of a universal vaccine difficult. The design of immunogenic peptides using bioinformatics tools could be an interesting strategy to increase the success of vaccines. In this work, we used the predicted amino acid sequences of the neuraminidase (NA) and hemagglutinin (HA) proteins of different IAV subtypes to perform multiple alignments, epitope predictions, molecular dynamics simulations, and experimental validation. Peptide selection was based on the following criteria: promiscuity, protein surface exposure, and the degree of conservation among different medically relevant IAV strains. These peptides were tested using immunological assays to test their ability to induce production of antibodies against IAV. We immunized rabbits and mice and measured the levels of IgG and IgA antibodies in serum samples and nasal washes. Rabbit antibodies against the peptides P11 and P14 (both of which are hybrids of NA and HA) recognized HA from both group 1 (H1, H2, and H5) and group 2 (H3 and H7) IAV and also recognized the purified NA protein from the viral stock (influenza A Puerto Rico/916/34). IgG antibodies from rabbits immunized with P11 and P14 were capable of recognizing viral particles and inhibited virus hemagglutination. Additionally, intranasal immunization of mice with P11 and P14 induced specific IgG and IgA antibodies in serum and nasal mucosa, respectively. Interestingly, the IgG antibodies were found to have neutralizing capability. In conclusion, the peptides designed through in silico studies were validated in experimental assays.

Testosterone induces hyporesponsiveness by interfering with IP3 receptors in guinea pig airway smooth muscle.

Asthma symptoms have been associated with sex steroids. During childhood, this illness seems more frequent in boys than in girls and this tendency reverts in puberty when it is more severe in women. Testosterone (TES), at supraphysiological concentrations, relaxed pre-contracted airway smooth muscle, but its effects at physiological concentrations have not been thoroughly studied. We explored this possibility in guinea pig tracheal smooth muscle. In myocytes TES (10 nM) abolished carbachol (CCh)-induced intracellular Ca2+ concentration ([Ca2+]i) increment. Ca2+ responses to ATP were partially modified by TES while histamine's were not. These results indicate that inositol 1,4,5-trisphosphate (IP3) signaling pathway might be involved. Photolysis of caged-IP3 increased [Ca2+]i and TES abolished this effect. TES diminished reactivity of the smooth muscle to CCh and this effect was non-genomic since it was unchanged by flutamide. In tracheal smooth muscle, mRNA for each IP3 receptor (ITPR) isoform was found and, by immunofluorescence, ITPR1 and ITPR3 seems to be the main isoforms observed while ITPR2 was less prominent. Comparing the amino acid sequence of ITPR1 and the sequence of the TES binding site on the androgen receptor, we found that they share a short sequence. This domain could be responsible for the TES binding to the ITPR1 and probably for its blocking effect. We conclude that TES modifies ITPR1 function in airway smooth muscle, turning this tissue less reactive to contractile agonists that act through PLCß-IP3 signaling cascade. These results might be related to the low asthma prevalence in males from puberty to adulthood.

Atomistic Characterization of the First Step of Calcium Pump Activation Associated with Proton Countertransport.

The calcium pump [sarcoplasmic reticulum (SR) Ca(2+)-ATPase (SERCA)] transports Ca(2+) from the cytosol to the SR lumen at the expense of ATP hydrolysis and proton countertransport, thus playing a central role in Ca(2+) homeostasis and muscle contractility. Proton countertransport via deprotonation of transport site residue Glu309 is a critical first step in SERCA activation because it accelerates the E2-E1 structural transition. Previous studies have suggested that flipping of Glu309 toward the cytosol constitutes the primary mechanism for Glu309 deprotonation, but no conclusive data to support this hypothesis have been published. Therefore, we performed three independent 1 µs molecular dynamics simulations of the E2 state protonated at transport site residues Glu309, Glu771, and Glu908. Structural analysis and pKa calculations showed that Glu309 deprotonation occurs by an inward-to-outward side-chain transition. We also found that Glu309 deprotonation and proton countertransport occur through transient (~113 ps) water wires connecting Glu309 with the cytosol. Although both mechanisms are operational, we found that transient water wire formation, and not Glu309 flipping, is the primary mechanism for Glu309 deprotonation and translocation of protons to the cytosol. The outward-to-inward transition of protonated Glu309 and the presence of water wires suggest that protons from the cytosol might be passively transported to the lumen via Glu309. However, structural analysis indicates that passive SR proton leakage into the lumen unlikely occurs through Glu309 in the E2 state. These findings provide a time-resolved visualization of the first step in the molecular mechanism of SERCA activation and proton transport across the SR.

Microsecond Molecular Simulations Reveal a Transient Proton Pathway in the Calcium Pump.

The calcium pump sarcoplasmic reticulum Ca(2+)-ATPase (SERCA) counter-transports Ca(2+) and H(+) at the expense of ATP hydrolysis. SERCA uses separate proton and metal ion pathways during active transport to neutralize the highly charged transport site, thus preserving SERCA's structural stability during active Ca(2+) transport. Although separate metal ion and proton pathways have been identified during slow (millisecond) structural transitions of SERCA, the existence of simultaneous metal and proton pathways during fast (microsecond) structural transitions remains unknown. We have analyzed microsecond-long trajectories of E1·H(+)771, a protonated intermediate of the pump populated during SERCA regulation. We found a transiently established hydrophobic pore in the luminal side of the transmembrane helices 6, 8, and 9. This narrow (0.5-0.6 nm) pore connects the transport sites to the sarcoplasmic reticulum lumen through a chain of water molecules. Protein pKa calculations of the transport site residues and structural analysis of the water molecules showed that this pore is suitable for proton transport. This transient proton pathway ensures neutralization of the transport sites during the rapid structural transitions associated with regulation of the pump. We conclude that this transient proton pathway plays a central role in optimizing active Ca(2+) transport by SERCA. Our discovery provides insight into ion-exchange mechanisms through transient hydrophobic pores in P-type ATPases.

Atomic-level mechanisms for phospholamban regulation of the calcium pump.

We performed protein pKa calculations and molecular dynamics (MD) simulations of the calcium pump (sarcoplasmic reticulum Ca(2+)-ATPase (SERCA)) in complex with phospholamban (PLB). X-ray crystallography studies have suggested that PLB locks SERCA in a low-Ca(2+)-affinity E2 state that is incompatible with metal-ion binding, thereby blocking the conversion toward a high-Ca(2+)-affinity E1 state. Estimation of pKa values of the acidic residues in the transport sites indicates that at normal intracellular pH (7.1-7.2), PLB-bound SERCA populates an E1 state that is deprotonated at residues E309 and D800 yet protonated at residue E771. We performed three independent microsecond-long MD simulations to evaluate the structural dynamics of SERCA-PLB in a solution containing 100 mM K(+) and 3 mM Mg(2+). Principal component analysis showed that PLB-bound SERCA lies exclusively along the structural ensemble of the E1 state. We found that the transport sites of PLB-bound SERCA are completely exposed to the cytosol and that K(+) ions bind transiently (≤5 ns) and nonspecifically (nine different positions) to the two transport sites, with a total occupancy time of K(+) in the transport sites of 80%. We propose that PLB binding to SERCA populates a novel (to our knowledge) E1 intermediate, E1⋅H(+)771. This intermediate serves as a kinetic trap that controls headpiece dynamics and depresses the structural transitions necessary for Ca(2+)-dependent activation of SERCA. We conclude that PLB-mediated regulation of SERCA activity in the heart results from biochemical and structural transitions that occur primarily in the E1 state of the pump.

Inhibition of influenza A virus infection in vitro by peptides designed in silico.

Influenza A viruses are enveloped, segmented negative single-stranded RNA viruses, capable of causing severe human respiratory infections. Currently, only two types of drugs are used to treat influenza A infections, the M2 H(+) ion channel blockers (amantadine and rimantadine) and the neuraminidase inhibitors (NAI) (oseltamivir and zanamivir). Moreover, the emergence of drug-resistant influenza A virus strains has emphasized the need to develop new antiviral agents to complement or replace the existing drugs. Influenza A virus has on the surface a glycoprotein named hemagglutinin (HA) which due to its important role in the initial stage of infection: receptor binding and fusion activities of viral and endosomal membranes, is a potential target for new antiviral drugs. In this work we designed nine peptides using several bioinformatics tools. These peptides were derived from the HA1 and HA2 subunits of influenza A HA with the aim to inhibit influenza A virus infection. The peptides were synthetized and their antiviral activity was tested in vitro against several influenza A viral strains: Puerto Rico/916/34 (H1N1), (H1N1)pdm09, swine (H1N1) and avian (H5N2). We found these peptides were able to inhibit the influenza A viral strains tested, without showing any cytotoxic effect. By docking studies we found evidence that all the peptides were capable to bind to the viral HA, principally to important regions on the viral HA stalk, thus could prevent the HA conformational changes required to carry out its membranes fusion activity.