Title:  Immunological activity for a peptide of the limulus anti-LPS factor

United States Patent:  6,632,793

Issued:  October 14, 2003

Inventors:  Vallespi; Maribel Guerra (Havana, CU); Rosianz; Manuel de Jesus Arana (Havana, CU)

Assignee:  Centro de Ingenieria Genetica y Biotecnologia (Havana, CU)

Appl. No.:  691500

Filed:  October 18, 2000

Abstract

The present invention relates a novel immunological effect of one peptide from Limulus anti-LPS factor protein. The immunological effect are, (1) Induce an antiviral state in both the Hep-2 and MDBK cell lines, (2) Supernatant from human mononuclear cells stimulated with peptide Limulus anti-LPS factor (LALF) is able to induce antiviral effect on Hep-2 cell line by mean of IFN-.gamma. (3) LALF peptide is able to modulate the immune response in vitro and in vivo. The present invention can be used in therapeutic and/or prophylactic treatment regimens of humans and animals to enhance their immune responses, without stimulating the production of certain biochemical mediators (e.g., TNF-.alpha.) that can cause detrimental effects, such as fever and inflammation.

DETAILED DESCRIPTION OF INVENTION

The invention relates to a peptide comprising the amino acids_31-52 (SEQ ID NO: 1) from Limulus anti-LPS factor protein isolate from a horseshoe crab such as Limulus polyphemus which enhance a host's immune defense mechanisms to infection but not induce an inflammatory response.

This peptide has been shown to have an antiviral effect to protect the cells against the viral infection in vitro assay. Furthermore, supernatant from human mononuclear cells incubated with the peptide contain certain soluble mediator(s), such as IFN-.gamma., able to confer protection to the cells Hep-2 against future viral infections. Another hand, this peptide protects against a direct infection in vivo. These advantageous properties make the peptide of this invention useful in the prevention and treatment of infections because they selectively activate only those components of the immune system responsible for the initial response to infection, without stimulating the release of certain biochemical mediators that can cause adverse side effects. The peptide also lacks the toxicity common to many immunomodulators.

The peptide of this invention is synthesized using a solid phase procedure. Crude peptide was extracted with a 30% acetic acid solution in water, lyophilized and then purified by RP-HPLC. Molecular mass of purified peptide was verified using a JEOL JMS-HX110HF two sector mass spectrometer equipped with a FAB gun. The resulting preparation is non-antigenic, non-pyrogenic and is pharmaceutically acceptable for administration to animals and humans.

The peptide comprising the amino acids31-52 (SEQ ID NO: 1) from Limulus anti-LPS factor protein of this invention can be used as safe, effective, therapeutic and/or prophylactic agents, either alone or as adjuvants, to enhance the immune response in humans and animals. The peptide comprising the amino acids sequences disclosed herein selectively activate only those components that are responsible for the initial response to infection caused both bacterial or viral pathogens, without stimulating or priming the immune system to release certain biochemical mediators (e.g., TNF, IL-1) that can cause adverse side effects. As such, the present peptide can be used to prevent or treat infectious diseases caused both bacterial or viral pathogens in malnourished patients, patients undergoing surgery and bone marrow transplants, patients undergoing chemotherapy or radiotherapy, neutropenic patients, HIV-infected patients, trauma patients, bum patients, patients with chronic or resistant infections, all of who may have weakened immune systems. An immunocompromised individual is generally defined as a person who exhibits an attenuated or reduced ability to mount a normal cellular and/or humoral defense to challenge by infectious agents, e.g., viruses, bacteria, fungi and protozoa. A protein malnourished individual is generally defined as a person who has a serum albumin level of less than about 3.2 grams per deciliter (g/dl) and/or unintentional weight loss of greater than 10% of usual body weight.

More particularly, the method of the invention can be used to therapeutically or prophylactically treat animals or human who are at a heightened risk of infection due to imminent surgery, injury, illness, radiation or chemotherapy, or other such condition which deleteriously affects the immune system. The method is useful to treat patients who have a disease or disorder which causes the normal metabolic immune response to be reduced or depressed, such as HIV infection (AIDS). For example, the method can be used to pre-initiate the metabolic immune response in patients who are undergoing chemotherapy or radiation therapy, or who are at a heightened risk for developing secondary infections or post-operative complications because of a diseases, disorder or treatment resulting in a reduced ability to mobilize the body's normal metabolic responses to infection. Treatment with the peptide has been shown to be particularly effective in mobilizing the host's normal immune defenses, thereby engendering a measure of protection from infection in the treated host. It may be used in elderly persons, immunologically depressed by non infectious diseases as cardiovascular or chronical diseases. It is known that these persons are susceptible to viral pneumonia e.g. the grippe virus. It may also be effective in the treatment of pneumonia due cytomegalovirus (CMV), which are more important every day in persons having immunity deficiency, e.g. persons treated with immuno-suppressors as a result of an organ transplant. The treatment with this peptide could be particularly attractive in strengthening the immune response in patients with trauma or it may be administered during the so called post-operational acute phase, where viral infections are generally associated producing severe consequences, where the patients do not respond to the antimicrobial treatments. Both in persons with traumas, as during the acute post-operational phase, there is a deterioration of the immune response given mainly by a decrease of the activity of the macrophages and their inability to respond adequately during an infection (Berger et al., Clin. Chem. Acta. 1995, Vol. 239: 121-130. Therefore, the agents that stimulate the function of the macrophages, as the main mediators of the immune response, would be of great therapeutic and prophylactic value in infectious diseases of viral or bacterial origin. The treatment with the peptide has demonstrated that it is particularly effective in mobilizing the normal immune defenses of the host, producing an increase in the protection of the host to the infection.

In another embodiment the peptide of the invention can be administrate to patients' undergoing "immunoparalysis" (Randow et al. , J. Exp. Med. 1995, Vol. 181: 1887-1892.), defined as the reduction of the monocytic MHC class II, impaired monocytic antigen-presenting activity and their diminished ability to produce inflammatory cytokines. This phenomenon is called Compensatory Anti-inflammatory Responses Syndrome (CARS). During the late hypoinflammatory phase in sepsis monocytic stimulation is useful because restitute the deficient phenotype and function of monocytes from patients with "immunoparalysis".

The present peptide is generally administered to an animal or human in an amount sufficient to produce immune system enhancement. The mode of administration of the peptide can be oral, enteral, parenteral, intravenous, subcutaneous, intraperitoneal, or intramuscular. The form in which the composition will be administered (e.g., tablet, capsule, solution, emulsion) will depend upon the route by which it is administered. The quantity of the composition to be administered will be determined on an individual basis, and will be based at least in part on consideration of the severity of infection or injury in the patient, the patient's conditions or overall health, the patient's weight and the time available before surgery, chemotherapy or other high-risk treatment. In general, a single dose will preferably contain approximately 5 mg to 10 mg of peptide per kilogram of body weight. In general, the composition of the present invention can be administrated to an individual periodically as necessary to stimulate the individual's immune response.

An individual skilled in the medical arts will be able to determined the length of time during which the composition is administrated and the dosage, depending upon the physical condition of the patient and the disease or disorder being treated. As stated above, the composition may also be used as a preventative treatment to pre-initiate the normal metabolic defenses, which the body mobilizes against infections.

The peptide can be used for the prevention and treatment of infections caused by a broad spectrum of bacterial, fungal, viral and protozoan pathogens. The prophylactic administration of peptide to a person undergoing surgery, either preoperatively, intraoperatively and/or post-operatively, will reduce the incidence and severity of post-operative infections in both normal and high-risk patients. For example, in patients undergoing surgical procedures that are classified as contaminated or potentially contaminated (e.g., gastrointestinal surgery, hysterectomy, cesarean section, transurethral prostatectomy) and in patients in whom infection at the operative site would present a serious risk (e.g., prosthetic arthroplasty, cardiovascular surgery), concurrent initial therapy with an appropriate antibacterial agent and the present peptide preparation will reduce the incidence and severity of infectious complications.

In patients who are immunosuppressed, not only by disease (e.g., cancer, AIDS) but by courses of chemotherapy and/or radiotherapy, the prophylactic administration of the peptide preparation will be reduce the incidence of infections caused by a broad spectrum of opportunistic pathogens including many unusual bacteria, fungi and viruses.

In high risk patients (e.g., over age 65, diabetics, patients having cancer, malnutrition, renal disease, emphysema, dehydration, restricted mobility, etc.) hospitalization frequently is associated with a high incidence of serious nosocomial infection. Treatment with this peptide preparation may be started empirically before characterization, use of respirators, drainage tubes, intensive care units, prolonged hospitalizations, etc. to help prevent the infections that are commonly associated with these procedures. Concurrent therapy with antimicrobial agents and the peptide preparation is indicated for the treatment of chronic, severe, refractory, complex and difficult to treat infections.

The compositions administered in the method of the present invention can optionally include other components, in addition to the peptide. The other components that can be included in a particular composition are determined primarily by the manner in which the composition is to be administered. For example, a composition to be administered orally in tablet form can include, in addition to peptide, a filler (e g., lactose) a binder (e.g., carboxymethyl cellulose, gum arabic, gelatin), an adjuvant, a flavoring agent, a coloring agent and a coating material (e.g., wax or plasticizer). A composition to be administered in liquid form can include peptide and, optionally, an emulsifying agent, a flavoring agent and/or a coloring agent. A composition for parenteral administration can be mixed, dissolved or emulsified in water, sterile saline, PBS, dextrose or other biologically acceptable carrier. A composition for topical administration can be formulated into a gel, ointment, lotion, cream or other form in which the composition is capable of coating the side to be treated, e.g., wound site.

Compositions comprising peptide preparation can be administered topically to a wound site to stimulated and enhance wound healing and repair. Wounds due to ulcers, acne, viral infections, fungal infections or periodontal disease, among other, can be treated according to the methods of this invention to accelerate the healing process. Alternatively, peptide preparation can be injected into wound or afflicted area. In addition to wound repair, the composition can be used to treat infection associated therewith or the causative agents that result in the wound. A composition for topical administration can be formulated into a gel, ointment, lotion, cream or other form in which the composition is capable of coating the site to be treated, e.g., wound site. A typical dosage for wound will be from about 5 mg to about 10 mg.

In another embodiment this invention relates to hybrid polypeptides containing the mentioned peptide sequences wherein the preferred peptide sequences constitutes the N-terminus or the C-termninus of larger polypeptide chains in a way that maintain its ability to enhance the immune response and confers this ability to the hybrid polypeptide. One preferred hybrid polypeptide comprises a fusion of any of the preferred peptides and heavy chain regions of IgG.

This invention also relates to scaffold proteins that properly exposed of the mentioned peptide sequences in such a way that maintains or enhances their ability to enhance the immune response and confers this ability to the hybrid polypeptide. The term "scaffold proteins" as used herein refers to hybrid polypeptides, which include within their polypeptide chain one or more of the selected LALF sequences in a such a way that the inserted segment forms an exposed loop in the structure of the fused protein, or polypeptide.

Peptide half life in vivo and other pharmacological parameters could be improved with hybrid and scaffold polypeptides and proteins including the preferred peptide sequence. Also DNA sequences encoding the peptides, or encoding hybrid or scaffold proteins containing the peptide sequence could be inserted in proper vectors to be expressed in vivo for therapeutic purposes along with appropriated carriers, diluents, adjuvant or stabilizing solutions or chemicals.

The novel immunological activity from Limulus anti-LPS factor peptide disclosed herein enhances the non-specific defenses of mammalian mononuclear cells and significantly increases their ability to respond to an infectious challenge. In another hand, it does not result in increased body temperatures (e.g., fever) as has been reported with many non-specific stimulants of those defenses. This critical advantage of peptide preparation may lie in the natural profile of responses it mediates in white blood cells. It has been shown that the effect of the present invention selectively activates immune responses but does not directly stimulate or prime cytokine (e g., TNF) release from mononuclear cells, thus distinguishing from other immunostimulants.

Claim 1 of 1 Claim

What is claimed is:

1. A viral vaccine comprising a peptide or protein having the amino acid residues 31-52 (SEQ. ID NO.1) of Limulus polyphemus anti-LPS factor, wherein said peptide or protein is an adjuvant in said viral vaccine.

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