News Column

"Compositions and Methods for Inducing Immune Responses against Bacteria in the Genus Staphylococcus" in Patent Application Approval Process

August 5, 2014



By a News Reporter-Staff News Editor at Life Science Weekly -- A patent application by the inventors Schlievert, Patrick M. (Iowa City, IA); Peterson, Marnie L. (Golden Valley, MN), filed on March 16, 2012, was made available online on July 24, 2014, according to news reporting originating from Washington, D.C., by NewsRx correspondents (see also Patents).

This patent application has not been assigned to a company or institution.

The following quote was obtained by the news editors from the background information supplied by the inventors: "Staphylococcus aureus is considered the most significant cause of serious infectious diseases in the United States; this is likely to be true world-wide as well. See, Klevens et al., JAMA 298:1763-1771 (2007); and Lowy, N Engl J Med 339:520-532 (1998). Serious illnesses caused by the organism include highly fatal pneumonia, in which as many as 35,000 patients succumb each year, infectious endocarditis, where S. aureus is the cause of up to 20,000 cases (10,000 fatalities, and significant survivor strokes and metastatic abscesses due to microbial clumps seeding the brain and other organs), sepsis where the organism is the second leading cause of bloodstream infections (for example 800,000 post-surgical infections), and osteomyelitis (S. aureus is the cause of nearly all cases). Additionally, S. aureus has become highly antibiotic resistant, with both community-associated and hospital-associated methicillin-resistant S. aureus (MRSA) arising.

"There have been major efforts by the medical and scientific communities to develop vaccines against S. aureus. However, all of them resulted in failure to date. Thus, there is a need for a vaccine against S. aureus."

In addition to the background information obtained for this patent application, NewsRx journalists also obtained the inventors' summary information for this patent application: "As described herein, administration of compositions containing two or more staphylococcal toxoids to animal models of human staphylococcal pneumonia and infection endocarditis completely protected the animals from challenge with S. aureus. Compositions described herein can be used for active immunization against bacteria of the genus Staphylococcus. In addition, compositions described herein can be used for generating antibodies for use as, for example, passive immunotherapeutic agents. Without being bound to a particular mechanism, Staphylococcus produces exotoxins to facilitate the organism's ability to cause infection. Thus, the compositions described herein can be used to enhance a subject's immune response to two or more staphylococcal exotoxins such that activity of the exotoxins is neutralized in the subject. Specifically, the staphylococcal exotoxins, which are in the family of molecules known as superantigens, contain a human cell receptor interaction site on CD40.

"As described herein, non-toxic superantigen mutants can amplify immune responses to a second antigen, staphylococcal .beta.-toxin, by 10 to 100-fold, as well as other antigens such as HIV proteins or sheep erythrocytes. In addition, antibodies against TSST-1 toxoids are capable of neutralizing superantigenicity and capable of protecting rabbits from lethal challenge by native TSST-1. Patients with staphylococcal TSS do not develop neutralizing antibody responses to the superantigen TSST-1, and thus they remain susceptible to TSS recurrences. This effect results from immune dysfunction due to TSST-1, rather than genetic inability to recognize the superantigen as foreign. TSST-1 toxoids described herein stimulate protective immunity against native TSST-1 and function as adjuvants to amplify antibody responses to secondary antigens. This effect is not seen with use of wild-type TSST-1, which is more likely to result in antibody immunosuppression than in adjuvanticity.

"In one aspect, this document features compositions that include two or more staphylococcal toxoids, wherein the toxoids are selected from the group consisting of a toxic shock syndrome toxin-1 (TSST-1) toxoid, a staphylococcal enterotoxin B (SEB) toxoid, a staphylococcal enterotoxin C (SEC) toxoid, a staphylococcal enterotoxin-like X (SEL-X) toxoid, an alpha toxin toxoid, a beta toxin toxoid, and a gamma toxin toxoid. The TSST-1 toxoid can include a serine residue at position 31 and a proline residue at position 32. The TSST-1 toxoid can be a fusion protein (e.g., a fusion of residues 1 to 89 of human TSST-1 and residues 90 to 195 of ovine TSST-1). The TSST-1 toxoid can include an alanine at position 135. The TSST-1 toxoid can include an alanine at position 136. The SEB toxoid can include one or more of the following: an alanine residue at position 90, a valine residue at position 91, and an alanine residue at position 210. The SEC toxoid can be a SEC3 toxoid. The SEC toxoid can include an alanine residue at position 90 and/or an alanine residue at position 210. The alpha toxin toxoid can include a leucine residue at position 35. The beta toxin toxoid can include an asparagine at position 149 and/or an asparagine at position 288. In some embodiments, the composition includes three staphylococcal toxoids. In some embodiments, the composition includes four staphylococcal toxoids. In some embodiments, the composition includes five staphylococcal toxoids. In some embodiments, the composition includes a TSST-1 toxoid, an SEB toxoid, an SEC toxoid, an alpha toxoid, and a beta toxoid. Such a composition further can include an SEL-X toxoid and/or a gamma toxin toxoid. In any of the compositions described herein, the gamma toxin toxoid can be a single chain of the gamma Staphylococcus toxin (e.g., the B chain of the gamma Staphylococcus toxin). In some embodiments, the composition includes a SEC toxoid, a SEB toxoid, and an alpha toxoid.

"Any of the compositions described herein further can include an adjuvant (e.g., incomplete Freund's adjuvant, complete Freund's adjuvant, or an aluminum salt).

"This document also features a method for inducing an immune response to two or more staphylococcal exotoxins produced by a strain of Staphylococcus in a subject. The method includes administering to the subject an amount of a pharmaceutical composition effective to induce the immune response, the pharmaceutical composition including two or more staphylococcal toxoids, wherein the toxoids are selected from the group consisting of a TSST-1 toxoid, an SEB toxoid, an SEC toxoid, an SEL-X toxoid, an alpha toxin toxoid, and a beta toxin toxoid. The composition can be administered subcutaneously or intramuscularly. The method further can include determining if the blood of the subject contains antibodies having specific binding affinity for one or more of the staphylococcal toxoids. The method further comprising determining if the blood of the subject contains antibodies having specific binding affinity for one or more of the staphylococcal exotoxins. The strain of Staphylococcus can be methicillin-resistant or methicillin-sensitive. The strain can be an isolate of USA400, USA300, or USA200. Any of the compositions described herein can be used in the methods.

"In another aspect, this document features a composition that includes a TSST-1 toxoid, an SEB toxoid, an SEC toxoid, a staphylococcal alpha toxin toxoid, a staphylococcal beta toxin toxoid, and a single chain of the gamma staphylococcal toxin. The composition further can include an adjuvant (e.g., incomplete Freund's adjuvant, complete Freund's adjuvant, or an aluminum salt).

"In another aspect, this document features a composition that includes a TSST-1 toxoid, an SEC toxoid, a staphylococcal alpha toxin toxoid, a staphylococcal beta toxin toxoid, and a single chain of the gamma staphylococcal toxin. The composition further can include an adjuvant (e.g., incomplete Freund's adjuvant, complete Freund's adjuvant, or an aluminum salt). Such a composition further can include a SEB toxoid.

"In another aspect, this document features a composition that includes a TSST-1 toxoid, an SEC toxoid, and a staphylococcal alpha toxin toxoid. Such a composition further can include a SEB toxoid. The composition further can include an adjuvant (e.g., incomplete Freund's adjuvant, complete Freund's adjuvant, or an aluminum salt).

"Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the exemplary methods and materials are described below. All publications, patent applications, patents, Genbank.RTM. Accession Nos, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present application, including definitions, will control. The materials, methods, and examples are illustrative only and not intended to be limiting.

"Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.

DESCRIPTION OF DRAWINGS

"FIG. 1 is a bar graph depicting the four hour change in temperature in rabbits after challenge with toxoid mutants of toxic shock syndrome toxin-1 (TSST-1) (G31S/S32P and Huvine, 1000 .mu.g/kg) (10 rabbits per toxoid challenge) or wild-type TSST-1 (1 .mu.g/kg) (5 rabbits). At the 4 hour time point, all rabbits were challenged with 50 .mu.g/kg of lipopolysaccharide (LPS) ( 1/10 LD.sub.50) alone. There is a 10.sup.6-fold synergy between superantigens (TSST-1) and LPS in causing lethal TSS.

"FIG. 2 is bar graph depicting the four hour change in temperature in rabbits after challenge with toxoid mutant SEC Y90A (10 rabbits, 1000 .mu.g/kg) or wild-type SEC3 (5 rabbits, 1 .mu.g/kg). At the 4 hour time point, all rabbits were challenged with 50 .mu.g/kg of LPS ( 1/10 LD.sub.50) alone. There is a 10.sup.6-fold synergy between superantigens (SEC3) and LPS in causing lethal TSS.

"FIG. 3 is a graph depicting the temperature (.degree. C.) in rabbits after challenge with toxoid mutants of toxic shock syndrome toxin-1 (TSST-1) ((.quadrature.), G31S/S32P; (.diamond-solid.), H135A (.tangle-solidup.); and Q136A ( )) (5 rabbits per toxoid challenge) or wild-type TSST-1 (1 .mu.g/kg) (5 rabbits). At the 4 hour time point, just after taking the 4 hr temperatures, all rabbits were challenged with 100 .mu.g/kg of LPS alone. Alive/Total refers to the number of animals that survived as measured 48 hr post LPS injection.

"FIG. 4 is a graph showing the superantigenicity of TSST-1 toxoid mutants (G31S/S32P [filled squares], Huvine [filled triangles]) as measured by incorporation of .sup.3H-thymidine into DNA of proliferating peripheral blood mononuclear cells (PBMCs). Toxoid and wild-type TSST-1 doses ranged from 10 .mu.g/well to 0.000001 .mu.g/well.

"FIG. 5 is a graph showing the superantigenicity.+-.standard deviation of TSST-1 (.quadrature.), G31S/S32P (.diamond-solid.), H135A (.tangle-solidup.), and Q136A ( ) for rabbit splenocytes in a 4-day assay. Rabbit splenocytes (2.times.10.sup.5/well) were incubated with TSST-1 and mutants for 3 days, and then 1 .mu.Ci.sup.3H-thymidine per well added for 24 hr. DNA was harvested, and counts per minute determined as a measure of T cell proliferation.

"FIG. 6 is a bar graph indicating the number of alive rabbits (prior immunization with a toxoid or non-immunized) after challenge with wild-type TSST-1. Prior immunization with G31S/S32P or Huvine toxoids protected rabbits from TSST-1 lethality. Animals were monitored 15 days for health.

"FIG. 7 is a bar graph indicating the number of alive rabbits (prior immunization with a toxoid or non-immunized) after challenge with wild-type SEC3. Prior immunization with SEC3 Y90A protected rabbits from SEC3 lethality. Animals were monitored 15 days for health.

"FIG. 8 is a bar graph comparing the superantigenicity of TSST-1 (1 .mu.g/well), in pooled rabbit sera from non-immune animals versus animals hyperimmune to TSST-1 mutants G31S/S32P, H135A, and Q136A, as tested in a 4 day assay with rabbit splenocytes. Splenocytes were incubated with designated dilutions of sera+TSST-1 for 3 days, and then 1 .mu.Ci.sup.3H-thymidine was added for 24 hr. DNA was harvested and counts/min determined as a measure of lymphocyte proliferation. Counts/min splenocytes+TSST-1=110,801.+-.8647. Counts/min splenocytes alone=7248.+-.1164.

"FIG. 9A is a line graph showing the number of pre-immunized or non-immunized rabbits alive after challenge with wild-type USA200 MNPE (for those immunized with G31S/S32P, G31S/S32P+Alpha, or Alpha alone) or USA400 MW2 (for those immunized with Y90A and Y90A+Alpha toxoid) intra-bronchially with 2.times.10.sup.9 bacteria per animal. The animals were monitored for 7 days for health. Prior immunization with G31S/S32P, Y90A.+-.Alpha toxoid protected rabbits from lethal pneumonia.

"FIG. 9B is a line graph showing the number of pre-immunized or non-immunized rabbits alive after challenge with wild-type USA200 MNPE. Rabbits (11/group) were immunized three times with antigens, TSST-1 (G31S/S32P)+SEC+.alpha.-toxin (H35L or wild-type) as a cocktail (.quadrature.) or .alpha.-toxin (H35L) alone (.diamond-solid.), or remained non-immunized (.tangle-solidup.). Antigens were emulsified in incomplete adjuvant and immune animals plus non-immune control animals challenged intrapulmonary with 2.times.10.sup.9 S. aureus MNPE. Rabbits immunized against TSST-1 (G31S/S32P)+SEC+.alpha.-toxin (H35L or wild-type) were significantly protected from lethality compared to non-vaccinated animals or animals vaccinated against .alpha.-toxin (H35L or wild-type) alone (p

"FIG. 10A is a line graph showing the number of pre-immunized or non-immunized rabbits (4-5 per group) alive after challenge with wild-type USA200 MNPE IV with 2.times.10.sup.8 bacteria per animal. The pre-immunized animals were immunized with G31S/S32P+Y90A+Alpha+Beta+Gamma or alpha toxoid alone. The health of the rabbits was monitored for 4 days.

"FIG. 10B is a line graph showing the number of pre-immunized or non-immunized rabbits (4-5 per group) alive after challenge with wild-type USA200 MNPE IV with 2.times.10.sup.8 bacteria per animal Rabbits were immunized three times with TSST-1 (G31S/S32P), SEC, .alpha.-toxin (H35L), .beta.-toxin, and .gamma.-toxin (.quadrature.) or .alpha.-toxin (H35L) alone (.diamond-solid.), or remained non-vaccinated (.tangle-solidup.). Challenge organism was intravenous USA200 S. aureus MNPE (2.times.10.sup.8/2 ml volume in PBS)

"FIG. 11 is a bar graph showing the antibody titer of Dutch-belted rabbits (3/group) in response to immunization with S. aureus .beta.-toxin (beta) alone and in combination with two toxoids of TSST-1: TSST-1 (Q136A) and TSST-1 (G31S/S32P). Immune responses in the presence of TSST-1 mutants, compared to responses to .beta.-toxin alone were significantly different (p

"FIG. 12 is a bar graph showing IL-8 production (pg/mL) from HVECs treated with TSST-1 and monoclonal antibodies against CD40 that neutralize interaction with T cell CD40 ligand. Monoclonal antibodies against CD40 alone (xCD40; 20 .mu.l undiluted), TSST-1 alone (100 .mu.g/ml), isotype-matched monoclonal antibodies against streptococcal pyrogenic exotoxin A (xSPEA) and monoclonal antibodies against CD40+TSST-1, and monoclonal antibodies against streptococcal pyrogenic exotoxin+TSST-1 were incubated with confluent HVECs in 96 well microtiter plates in triplicate for 6 hr. Subsequently IL-8 production was measured by ELISA. Bars represent standard error of the means.

"FIG. 13 is a bar graph depicting the temperature (.degree. C.) and survival of rabbits immunized or not immunized against alpha toxin and the respective superantigen (SEC or SEC) produced by the challenge CA-MRSA strain when organisms were administered intra-pulmonary. Fevers were measured with the use of rectal thermometers prior to infection and on day 1 post-infection. Deaths were recorded over a 7 day time-period."

URL and more information on this patent application, see: Schlievert, Patrick M.; Peterson, Marnie L. Compositions and Methods for Inducing Immune Responses against Bacteria in the Genus Staphylococcus. Filed March 16, 2012 and posted July 24, 2014. Patent URL: http://appft.uspto.gov/netacgi/nph-Parser?Sect1=PTO2&Sect2=HITOFF&u=%2Fnetahtml%2FPTO%2Fsearch-adv.html&r=2539&p=51&f=G&l=50&d=PG01&S1=20140717.PD.&OS=PD/20140717&RS=PD/20140717

Keywords for this news article include: Alanine, Patents, Aluminum, Exotoxins, Bacillales, Immunology, Amino Acids, Enterotoxins, Legal Issues, Light Metals, Superantigens, Blood Proteins, Staphylococcus, Immunoglobulins, Serum Globulins, Biological Toxins, Staphylococcaceae, Biological Factors, Gram-Positive Cocci, Monoclonal Antibodies, Gram-Positive Bacteria.

Our reports deliver fact-based news of research and discoveries from around the world. Copyright 2014, NewsRx LLC


For more stories covering the world of technology, please see HispanicBusiness' Tech Channel



Source: Life Science Weekly


Story Tools






HispanicBusiness.com Facebook Linkedin Twitter RSS Feed Email Alerts & Newsletters