José V. Torres

RNAIII inhibiting peptide (RIP), a global inhibitor of Staphylococcus aureus pathogenesis: Structure and function analysis

Staphylococcus aureus are gram-positive bacteria that can cause serious diseases in humans and animals. S. aureus infections can be prevented by the heptapeptide RNAIII inhibiting peptide (RIP). RIP was originally isolated from culture supernatants of coagulase negative staphylococci presumed to be S. xylosus. The sequence of RIP was identified as YSPXTNF. Native RIP and its synthetic analogue YSPWTNF have been shown to be effective inhibitors of diseases caused by various strains of S. aureus, including, cellulitis, keratitis, septic arthritis, osteomylitis and mastitis. RIP is therefore considered to be a global inhibitor of S. aureus. We show here that: 1) the amide form of RIP (YSPWTNF-NH2) is highly stable and is therefore the one recommended for use. 2) RIP inhibits S. aureus pathogenesis by inhibiting the synthesis of both agr transcripts RNAII and RNAIII. 3) Although RIP inhibits agr, it also reduces bacterial adherence to mammalian cells and to plastic (tested on HEp2 cells and on polystyrene by fluorescence and atomic force microscopy), suggesting that RIP can be used safely as a therapeutic molecule. 4) RIP derivatives were designed and tested for their ability to inhibit RNAIII in vitro and cellulitis in vivo. Not all peptides that inhibited RNAIII also inhibited an infection in vivo, indicating that studies must be carried out in vivo before considering a peptide to be of therapeutic potential. 5) The RIP derivative containing Lysine and Isoleucine at positions 2 and 4, respectively, inhibited S. aureus infections in vivo (tested on cellulitis), suggesting that both RIP YSPWTNF and its derivative YKPITNF are effective inhibitors of infections caused by S. aureus.

Prevention of diseases caused by Staphylococcus aureus using the peptide RIP.

Staphylococcus aureus causes many diseases including cellulitis, keratitis, osteomyelitis, septic arthritis and mastitis. The heptapeptide RIP has been shown to prevent cellulitis in mice, which was induced by S. aureus strain Smith diffuse. Here we show that RIP can also significantly reduce the overall pathology and delay the onset of disease symptoms in several other models of S. aureus infections, including: keratitis (tested in rabbits against S. aureus 8325-4), osteomyelitis (tested in rabbits against S. aureus MS), mastitis (tested in cows against S. aureus Newbould 305, AE-1, and environmental infections) and septic arthritis (tested in mice against S. aureus LS-1). These findings substantiate that RIP is not strain specific in its inhibitory activity and that RIP is an effective inhibitor of bacterial pathology at multiple body sites following diverse routes and doses of administration. These findings strongly evidence the potential value of RIP as a chemotherapeutic agent.

Morphine Induces Gene Expression of CCR5 in Human CEM x174 Lymphocytes

All HIV-1 strains studied to date use CCR5, CXCR4, or both receptors to enter cells. Simian immunodeficiency virus (SIV) infection of non-human primates has served as a useful model for understanding AIDS pathogenesis in humans. Research on several genetically divergent SIV isolates has revealed that SIV uses CCR5, and not CXCR4, for entry. CEM x174, a human lymphoid cell line, has been routinely used to cultivate and maintain various SIV strains. However, questions have arisen about how CEM x174, which reportedly was unable to express detectable amounts of CCR5 transcripts, efficiently supports the growth of SIV. In searching for an answer, we resorted to a sensitive competitive reverse transcriptase-polymerase chain reaction procedure in an attempt to detect as well as quantify the amount of CCR5 expression. Here we present our findings, which indicate that CEM x174 indeed expresses CCR5 and that the amount of CCR5 is increased in cells pretreated with morphine. These results correlate well with our previous observations that morphine treatment causes CEM x174 cells to be more susceptible to SIV infection. Similar morphine effect was not observed on CEM x174 cells infected with simian retroviruses, which do not depend on CCR5 for entry. These findings suggest a plausible mechanism whereby opiate drug users render themselves more susceptible to HIV infection, thereby explaining the vast prevalence of HIV infection among endemic drug use populations.

Methadone induces CCR5 and promotes AIDS virus infection

Methadone, a regimen for the treatment of opioid dependency, was found to induce the expression of CCR5, a co‐receptor for human immunodeficiency virus (HIV)/simian form of HIV (SIV) entry, on human CEM x174 lymphocytes. Both CCR5 mRNA and protein were elevated in methadone‐treated cells. A concomitant increase of mu opioid receptors was also observed. Upon methadone exposure, SIVmac239‐infected CEM x174 cells released greater amounts of virus particles as revealed by both the number of syncytia formation and reverse transcriptase activities. Similar methadone effect was not observed on CEM x174 cells infected with other simian retroviruses that do not depend on CCR5 for cellular entry. These studies raise concerns considering methadone as an innocuous morphine substitute.

Induction of Broad Cross-Subtype-Specific HIV-1 Immune Responses by a Novel Multivalent HIV-1 Peptide Vaccine in Cynomolgus Macaques

One of the major obstacles in the design of an effective vaccine against HIV-1 is its antigenic variation, which results in viral escape from the immune system. Through a bioinformatics approach, we developed an innovative multivalent HIV-1 vaccine comprised of a pool of 176 lipidated and nonlipidated peptides representing variable regions of Env and Gag proteins. The potency and breadth of the candidate vaccine against a panel of HIV-1 subtypes was evaluated in nonhuman primate (cynomolgus macaques) and humanized mouse (HLA-A2.1) models. The results demonstrate strong immunogenicity with both breadth (humoral and cellular immunity) and depth (immune recognition of widely divergent viral sequences) against heterologous HIV-1 subtypes A–F.

Trichomonas vaginalis: In vitro attachment and internalization of HIV-1 and HIV-1-infected lymphocytes

Abstract Sexually transmitted diseases (STDs) caused by bacteria and protozoa play an important role in the epidemiology of human immunodeficiency virus (HIV-1) infection. Human trichomoniasis, produced by the protozoan parasite Trichomonas vaginalis, is one of the most common STDs, and is a cause of mucosal lesions in the urogenital tract, which may increase the risk for HIV infection. However, there are no reports concerning the outcome of in vitro interactions between HIV particles and trichomonads. Therefore, we incubated T. vaginalis with three subtypes of HIV-1 (A, B, and D), as well as with HIV-1-infected lymphocytes, and analyzed the interactions with immunofluorescence microscopy and transmission electron microscopy. Our results demonstrated that HIV-1 particles attach and are incorporated into T. vaginalis through endocytic vesicles and are degraded within cytoplasmic vacuoles in approximately 48 h. There was no ultrastructural evidence of HIV-1 replication in trichomonads. These results demonstrated that trichomonads may internalize and harbor HIV-1 particles for short periods of time. In addition, under in vitro conditions, T. vaginalis ingests and digests HIV-1-infected lymphocytes.

Immunogenicity of a vaccine preparation representing the variable regions of the HIV type 1 envelope glycoprotein.

Variability of the major antigenic sites of the envelope glycoprotein of HIV-1 constitutes a major problem in the formulation of effective vaccines. We have prepared a synthetic peptide vaccine that represents the major hypervariable epitopes (V1 through V5) of the clade B HIV-1 envelope glycoprotein (gp120). We refer to this preparation as variable epitope immunogen or VEI vaccine. This construct takes into consideration the type and frequency of amino acid substitutions found at each epitope during the evolution of the virus in individual patients and in the target population. Immunization of mice, rabbits, and rhesus macaques with the VEI vaccine resulted in the induction of long-lasting, high-titered HIV-1 antibodies, including antibodies that neutralize primary isolates. We also documented lymphocyte proliferative responses to the VEI vaccine, its individual components, analogs, and subtype-specific peptides representing the major hypervariable regions of HIV-1 gp120. Delayed-type hypersensitivity responses to these antigens were also demonstrated in mice. Our results show that this vaccine is highly immunogenic and safe in animals. Our data suggest that this formulation could become an important component of combination vaccine approaches against HIV-1 and other antigenically variable pathogens.

Effect of Human Immunodeficiency Virus Type 1 on Intracellular Activation and Superoxide Production by Neutrophils

The immunopathogenesis of AIDS is associated with the development of opportunistic infections by intracellular pathogens that can invade and reproduce freely because of impaired cellular functions. Neutrophils from asymptomatic human immunodeficiency virus (HIV) type 1-infected persons and from symptomatic patients with AIDS were found to retain normal phagocytosis activity while producing significantly less superoxide than neutrophils from HIV-1-negative subjects, when stimulated through Fc receptors or protein kinase C. After priming with a synthetic HIV-1 envelope peptide and stimulation via the Fc receptor, the neutrophils from HIV-1-negative controls had suppressed superoxide production, reduced phosphorylation of two unidentified cellular proteins, and increased expression of a third phosphoprotein. These results suggest that HIV-1 can produce direct functional damage of neutrophils through binding of envelope components to the cell membrane.

Immunological and chemical analysis of proteins from Eisenia foetida earthworm

Previous studies have demonstrated the fibrinolitic and antibacterial activities of proteins from the earthworm Eisenia foetida (Ef) but their use as a food supplement for animals and humans is still being considered. We studied proteins from this worm in order to determine chemical composition (protein, fat and heavy metal levels), electrophoretic profile in SDS-PAGE and 2D gels, induction of humoral immune response in mice and toxicity in a human cell line. Chemical analysis revealed 61.8, 11.3 and 8.7 g% of proteins, fat and ash, respectively. Levels of heavy metals were low and similar to tunny fish. Sixteen bands were obtained by SDS-PAGE with a molecular weigh range of 6.9–205 kDa while twenty-three spots were observed following 2D gel electrophoresis. After three immunizations with Eisenia foetida lysate, high titer antibodies were produced without causing immediate hypersensitivity reactions. Good cell viability was observed when Ef proteins were added to a human cell line. These results suggest that Ef proteins are safe for feeding animals intended for human consumption.

Antibodies from HIV-positive and AIDS patients bind to an HIV envelope multivalent vaccine.

A major problem impeding development of an effective HIV vaccine is the rapid antigenic variability that is characteristic of several envelope glycoprotein epitopes. Frequent mutations alter the composition of the most immunogenic regions of the envelope glycoprotein. We have prepared a synthetic immunogen representing the evolution of the major hypervariable epitopes on the envelope glycoprotein (gp120) of HIV-1. Five synthetic constructs, representing each of the HIV-1 gp120 hypervariable epitopes were tested for recognition by antibodies from patients infected with HIV-1 from different geographic regions worldwide. An HIV-1 human plasma panel provided a representation of the antibodies recognizing subtype-specific epitope sequences prevalent at different parts of the world. The vaccine construct was recognized by antibodies from HIV-1-positive individuals infected with subtypes A, B, C, D, E, and F. Antibodies in pooled HIV-1 patient sera from San Francisco also recognized all five constructs. This complex immunogen was recognized by antibodies in sera from individual HIV-1-positive and AIDS patients from Puerto Rico and Canada, with a strong binding to the complete vaccine and the V3 component. Altogether, our results demonstrate that antibodies from seropositive patients infected with different HIV-1 clades recognize and bind to the HIV hypervariable epitope construct vaccine preparation and its individual components.